Recombinant fusion proteins and libraries from immune cell repertoires

ABSTRACT

Disclosed herein are methods and compositions for generating a repertoire of recombinant fusion polypeptides from immune cells, and uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/159,705,filed May 19, 2016, which is a continuation of U.S. application Ser. No.14/734,953, filed Jun. 9, 2015, now U.S. Pat. No. 9,422,547, thedisclosures of which are hereby incorporated by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 16, 2017, isnamed 37055US_CRF_sequencelisting.txt and is 34,085 bytes in size.

FIELD OF THE INVENTION

The invention relates to methods and compositions for proteinengineering for use in biomedical applications.

BACKGROUND OF THE INVENTION

Antibody therapeutics are increasingly used by pharmaceutical companiesto treat intractable diseases such as cancer (Carter 2006 Nature ReviewsImmunology 6:343-357). However, the process of antibody drug discoveryis expensive and tedious, and has proceeded by identification of anantigen, and then the isolation and production of antibodies withactivity against the antigen. Furthermore, affinity selection andexpression of a limited number of antibodies from this selection hasprovided a mechanism of treatment that is narrower than that provided bythe body. However, artificial generation of a representative immunerepertoire from an individual with cognate paired heavy and light chainimmunoglobulin or T cell receptors has not been achieved.

Individuals that have been exposed to disease naturally produceantibodies against antigens associated with that disease. Therefore,what is needed are improved methods of high throughput generation ofrecombinant fusion proteins comprising both heavy and light chainvariable domains so it is possible to use natural immune repertoires intreatment and pharmaceutical discovery and development.

SUMMARY OF THE INVENTION

Disclosed herein are methods and compositions of generating arecombinant fusion polypeptide. In an embodiment, disclosed herein is amethod for preparing a recombinant immunoglobulin library, comprising:providing primary immune cells from at least one mammalian donor;isolating in a plurality of monodisperse droplets single immune cellsfrom said primary immune cells; generating a plurality of recombinantfusion polynucleotides each comprising a first polynucleotide encoding aheavy chain variable domain and a second polynucleotide encoding a lightchain variable domain, wherein said heavy chain variable domain andlight chain variable domain on each of said plurality of recombinantfusion polynucleotides are a cognate pair from one of said isolatedprimary immune cells, wherein each of said plurality of recombinantfusion polynucleotides further comprise a linker polynucleotide linkingsaid first and second polynucleotides; circularizing each of saidplurality of linear recombinant fusion polynucleotides; and inserting athird polynucleotide comprising a sequence encoding a promoter and asequence encoding a constant region between said first and secondpolynucleotide in each of said circularized recombinant fusionpolynucleotides, thereby generating at least 1,000 unique recombinantimmunoglobulin expression constructs, thereby generating a recombinantimmunoglobulin library.

In an embodiment, the method further comprises inserting said at least1,000 recombinant immunoglobulin expression constructs into a pluralityof host cells; and expressing said at least 1,000 recombinantimmunoglobulin expression constructs in said plurality of host cells,thereby generating a recombinant immunoglobulin library comprising atleast 1,000 unique recombinant immunoglobulins, wherein each of said atleast 1,000 unique recombinant immunoglobulins comprises a linked heavychain and light chain variable domain cognate pair from a singleisolated cell, thereby generating a recombinant immunoglobulin library.

In an embodiment, the recombinant immunoglobulin library encodes least10,000, 100,000, or 1,000,000 unique recombinant immunoglobulinproteins. In an embodiment, the at least one mammalian donor is selectedbased on a medical criterion. In an embodiment, the medical criterioncomprises a heightened immune response to hepatitis B, rabies, tetanustoxin, varicella-zoster, cytomegalovirus, or pneumococcus.

In an embodiment, the at least one mammalian donor was exposed to anantigen of interest or a pathogen. In an embodiment, the antigen ofinterest is a tumor antigen selected from the group consisting of: CD19,CD20, CD22, CD52, epidermal growth factor receptor (EGFR), humanepidermal growth factor receptor 2 (HER2), tumor necrosis factorreceptor superfamily, member 10a (TRAILR1), receptor activator ofnuclear factor kappa-B ligand (RANKL), insulin-like growth factor 1receptor (IGF1R), epithelial cell adhesion molecule (EpCAM),carcinoembryonic antigen (CEA), and mucin 5AC. In an embodiment, theantigen of interest is an autoimmune antigen selected from the groupconsisting of: thrombin, nicotinic acetylcholine receptor,thyroglobulin, TSH receptor, glutamate decarboxylase,phospholipase-A2-receptor (PLA2R), and muscle specific kinase (MUSK).

In an embodiment, the at least one mammalian donor was immunized againsta pathogen. In an embodiment, the at least one mammalian donor has anautoimmune disease or cancer. In an embodiment, the at least onemammalian donor has idiopathic (immune) thrombocytopenic purpura (ITP),Kawasaki's vasculitis, B cell chronic lymphocytic leukemia (CLL), orprimary immunodeficiencies. In an embodiment, the recombinantimmunoglobulin expression construct is an expression vector.

Also provided herein is a method for preparing a plurality ofrecombinant immunoglobulin expression constructs, comprising: providinga plurality of recombinant fusion polynucleotides each comprising afirst polynucleotide encoding a first variable domain, a secondpolynucleotide encoding a second variable domain, and a linkerpolynucleotide linking the first and second polynucleotides; expressinga plurality of recombinant fusion polypeptides encoded by said pluralityof recombinant fusion polynucleotides to display said plurality ofrecombinant fusion polypeptides on a plurality of surfaces; enrichingsaid plurality of recombinant fusion polynucleotides for binding to anantigen by exposing said plurality of surfaces to said antigen andselecting based on binding between said antigen and each of saidplurality of recombinant fusion polypeptides; circularizing one or moreof said enriched recombinant fusion polynucleotides; and inserting athird polynucleotide comprising a sequence encoding a promoter and asequence encoding a constant region between said first and secondpolynucleotide in each of said circularized enriched recombinant fusionpolynucleotides, thereby generating a plurality of recombinantimmunoglobulin expression constructs.

In an embodiment, the method further comprises inserting said pluralityof recombinant immunoglobulin expression constructs into a plurality ofhost cells; and expressing said plurality of recombinant immunoglobulinexpression construct in said plurality of host cells, thereby generatinga recombinant immunoglobulin library, wherein each recombinantimmunoglobulin comprises a linked heavy chain and light chain variabledomain cognate pair from a single isolated cell.

In an embodiment, the first variable domain is from an immunoglobulinheavy chain, and said second variable domain is from an immunoglobulinlight chain. In an embodiment, the plurality of recombinantimmunoglobulin expression constructs comprises at least 1,000, 10,000,or 100,000 unique cognate pairs of heavy chain and light chain encodingsequences.

Also provided herein is a method of generating an immunoglobulinlibrary, comprising: providing a plurality of circularizedpolynucleotide constructs, each comprising a first polynucleotide, asecond polynucleotide, and a linker polynucleotide linking the first andsecond polynucleotides, wherein said first polynucleotide comprises aregion encoding a first variable domain from a single isolated cell,wherein said second polynucleotide comprises a region encoding a secondvariable domain from said single isolated cell, and wherein each of saidplurality of circularized polynucleotide constructs comprises a cognatepair of linked first and second variable domains from a single isolatedcell; inserting a third polynucleotide comprising a sequence encoding apromoter and a sequence encoding a constant region between said firstand second polynucleotide in each of said circularized recombinantfusion polynucleotides, thereby generating a plurality of recombinantimmunoglobulin expression construct; inserting said plurality ofrecombinant immunoglobulin expression constructs into a plurality ofhost cells; and expressing said plurality of recombinant immunoglobulinexpression construct in said plurality of host cells, thereby generatinga recombinant immunoglobulin library comprising a plurality ofrecombinant immunoglobulins, wherein each of said plurality ofrecombinant immunoglobulins comprises a linked heavy chain variabledomain and light chain variable domain cognate pair from a singleisolated cell.

In an embodiment, the insertion of said third polynucleotide isperformed in parallel for said plurality of circularized polynucleotideconstructs. In an embodiment, at least one recombinant immunoglobulinfrom said recombinant immunoglobulin library specifically binds to aStreptococcus pneumonia epitope, a Hemophilis influenza epitope, or aKlebsiella pneumonia epitope. In an embodiment, the first variabledomain is from an immunoglobulin heavy chain, and said second variabledomain is from an immunoglobulin light chain. In an embodiment, theplurality of recombinant fusion protein expression constructs areexpression vectors. In an embodiment, the insertion of said thirdpolynucleotide comprises a method selected from the group consisting of:Gibson assembly, site-specific digestion and ligation, and targetedrecombination.

Also provided herein is a composition comprising a pool of at least10,000 monodisperse aqueous droplets in an oil solution, wherein saidaqueous droplets each have a diameter of between 1 micron and 200microns, wherein each of said aqueous droplets have an outer boundarycomprising a surfactant, and wherein a plurality of said at least 10,000monodisperse aqueous droplets comprise a first probe comprising a firstpolynucleotide having a length of between 15 and 120 nucleotides,wherein said first polynucleotide is complementary to a firstsubsequence of a polynucleotide encoding a constant domain or poly(A)tail of an antibody or T cell receptor, and a second probe comprising asecond polynucleotide having a length of between 15 and 120 nucleotides,wherein said second polynucleotide is complementary to a secondsubsequence of a polynucleotide encoding a constant domain or poly(A)tail of an antibody or T cell receptor; wherein said first and secondprobes are attached to at least one particle.

In an embodiment, the first and second nucleic acid probes are bound toa particle. In an embodiment, the particle is a bead comprising agarose,glass, chemical polymers, or magnetic materials. In an embodiment, theprobes comprise biotin and wherein said particle comprises streptavidinbound to the surface of the particle. In an embodiment, the compositioncomprises a covalent bond between said first probe or said second probeand said particle. In an embodiment, each of said plurality ofmonodisperse aqueous droplets further comprise reagents for overlapextension RT-PCR. In an embodiment, the first probe is bound to apolynucleotide encoding a light chain variable domain from a singleisolated cell, and wherein said second probe is bound to apolynucleotide encoding a heavy chain variable domain from said singleisolated cell.

Also provided herein is a method for preparing a recombinant fusionprotein expression construct, comprising: providing a linearpolynucleotide construct comprising a first polynucleotide encoding afirst variable domain, a second polynucleotide encoding a secondvariable domain, and a linker polynucleotide linking the first andsecond polynucleotides; circularizing said linear polynucleotide, sothat said first and second variable domain are connected by said linkerpolynucleotide and a second polynucleotide; and inserting a thirdpolynucleotide encoding a transcriptional modulator, a translationalmodifier, a constant domain, or an immune effector into the spacerpolynucleotide, thereby generating a recombinant fusion proteinexpression construct.

In an embodiment, the first and second variable domains are from asingle cell. In an embodiment, the first variable domain is from animmunoglobulin heavy chain, and said second variable domain is from animmunoglobulin light chain. In an embodiment, the recombinant fusionprotein expression construct is an expression vector.

Also provided herein is a method of generating a plurality ofcircularized polynucleotides, comprising: providing primary immune cellsfrom at least one mammalian donor; isolating single immune cells fromsaid primary immune cells in a plurality of reaction vessels; generatinga plurality of linear recombinant fusion polynucleotides each comprisinga first polynucleotide encoding a heavy chain variable domain and asecond polynucleotide encoding a light chain variable domain from eachisolated single immune cell, wherein each of said plurality of linearrecombinant fusion polynucleotides further comprise a linkerpolynucleotide linking said first and second polynucleotides; andcircularizing said linear recombinant fusion polynucleotide.

Disclosed herein are methods and compositions of generating arecombinant fusion polypeptide. In an embodiment, the method comprisesproviding, in a reaction vessel, a lysis solution and a first nucleicacid probe and a second nucleic acid probe, wherein said first andsecond nucleic acid probes are attached to the same or differentsubstrates, wherein said first nucleic acid probe is capable ofhybridizing to a complementary region on first polynucleotide comprisinga first variable domain, and wherein said second nucleic acid probe iscapable of hybridizing to a complementary region on a second targetpolynucleotide comprising a second variable domain, wherein said firsttarget polynucleotide comprises a first variable domain, and said secondtarget polynucleotide comprises a second variable domain adding one ormore immune cells to said reaction vessel, thereby lysing said one ormore immune cells; hybridizing said first probe to said first targetpolynucleotide; hybridizing said second probe to said second targetpolynucleotide; and generating a recombinant fusion polynucleotidecomprising said first variable domain and said second variable domain.

In an embodiment, the method further comprises isolating said same ordifferent substrates from said lysis solution after hybridization insaid reaction vessel. In an embodiment, the method further comprisesexpressing said recombinant fusion polynucleotide in a host cell,thereby generating a recombinant fusion polypeptide. In an embodiment,the method further comprises purifying the recombinant fusionpolypeptide. In an embodiment, said recombinant fusion polypeptidecomprises a single chain variable fragment (scFv) or an antibodyfragment.

In an embodiment, the method further comprises said recombinant fusionpolypeptide is secreted by the host cell. In an embodiment, saidrecombinant fusion polypeptide comprises at least two unique variabledomains from the same antibody from said one or more immune cells. In anembodiment, said recombinant fusion polypeptide comprises at least twounique variable domains form the same T cell receptor from said one ormore immune cells. In an embodiment, said host cell is a yeast, insect,bacteria, or mammalian cell.

In an embodiment, the step of generating a recombinant fusionpolynucleotide comprises using overlap extension PCR to fuse and amplifythe first variable domain and the second variable domain. In anembodiment, the one or more immune cells is a subpopulation of antibodyor T-cell receptor producing cells. In an embodiment, the one or moreimmune cells expresses an antibody or T-cell receptor. In an embodiment,the reaction vessel is a droplet. In an embodiment, the droplet is anaqueous droplet in an oil solution, said aqueous droplet comprising anouter layer comprising a surfactant boundary. In an embodiment, theparticle is a bead. In an embodiment, the first nucleic acid probe andsaid second nucleic acid probe are each between 20 and 130 nucleotidesin length. In an embodiment, the first and second nucleic acid probeseach are complementary to a subsequence of the constant domain of anantibody or T cell receptor.

In an embodiment, the first nucleic acid probe attached to a particle,said second nucleic acid probe attached to a particle and said one ormore immune cells are encapsulated in a reaction vessel. In anembodiment, the one or more immune cells comprise an immune cellpopulation. In an embodiment, the first and second targetpolynucleotides each encode a portion of a different protein subunit.

In an embodiment, the recombinant fusion polynucleotide is generated byamplification of said first target polynucleotide and said second targetpolynucleotide and fusing said first and second amplified products. Inan embodiment, the amplification is performed using droplet PCR. In anembodiment, the first variable domain and said second variable domainare separated by less than 1,000, 900, 800, 700, 600, 500, 400, 300,200, 100, 80, 60, 40, or 20 nucleotides in the recombinant fusionpolynucleotide.

In an embodiment, the one or more immune cells comprise B cells orplasma cells. In an embodiment, the first variable domain encodes alight chain variable domain polypeptide, and said second variable domainencodes a heavy chain variable domain polypeptide. In an embodiment ofthe method, a linker polynucleotide is disposed between the firstvariable domain and the second variable domain in said recombinantfusion polynucleotide.

In an embodiment, the first probe comprises a polynucleotide sequencecomplementary to a portion of a light chain constant domain encodingpolynucleotide, and wherein said second probe comprises a polynucleotidesequence complementary to a portion of a heavy chain constant domainencoding polynucleotide. In an embodiment, the first probe and saidsecond probe comprise oligo(dT) polynucleotides capable of hybridizingto the poly(A) tail of mRNA.

Also provided herein is a method for preparing a recombinant fusionpolynucleotide protein expression construct, comprising: providing apolynucleotide construct comprising a first polynucleotide encoding afirst variable domain, a second polynucleotide encoding a secondvariable domain, and a spacer polynucleotide linking the first andsecond polynucleotides; and inserting a third polynucleotide encoding atranscriptional modulator, a translational modifier, a constant domain,or an immune effector into the spacer polynucleotide, thereby generatingsaid recombinant fusion polynucleotide protein expression construct.

In an embodiment, the variable domains are amplified from a single cellor subpopulation of cells. In an embodiment, the first variable domainis an immunoglobulin heavy chain, and said second variable domain is animmunoglobulin light chain. In an embodiment, the first variable domainis a T cell receptor alpha and said second variable domain is a T cellreceptor beta. In an embodiment, the recombinant fusion polynucleotideexpression recombinant fusion protein expression construct is anexpression vector. In an embodiment, the expression vector is a plasmidor a phagemid. In an embodiment, the insertion of said thirdpolynucleotide into said spacer polynucleotide comprises a methodselected from the group consisting of: Gibson assembly, site-specificdigestion and ligation, and targeted recombination.

Also provided herein is a recombinant fusion polynucleotide expressionrecombinant fusion protein expression construct comprising a firstpolynucleotide encoding a first variable domain, a second polynucleotideencoding a second variable domain, and a spacer polynucleotide linkingthe first and second polynucleotides, wherein said spacer polynucleotidecomprises a transcriptional modulator, a translational modifier, aconstant domain, or an immune effector. In an embodiment, the firstvariable domain and said second variable domain are from a single cellor subpopulation of cells. In an embodiment, the first variable domainis an immunoglobulin heavy chain, and said second variable domain is animmunoglobulin light chain. In an embodiment, the first variable domainis a T cell receptor alpha and said second variable domain is a T cellreceptor beta. In an embodiment, the recombinant fusion polynucleotideexpression recombinant fusion protein expression construct is anexpression vector. In an embodiment, the expression vector is a plasmidor a phagemid.

Also provided herein is a method of generating a recombinant fusionpolypeptide, comprising: inserting a recombinant fusion polynucleotideexpression recombinant fusion protein expression construct into a hostcell, wherein said recombinant fusion polynucleotide expressionrecombinant fusion protein expression construct comprises apolynucleotide encoding a first variable domain, a second polynucleotideencoding a second variable domain, and a spacer polynucleotide linkingthe first and second polynucleotides, wherein said spacer polynucleotidecomprises a transcriptional modulator, a translational modifier, aconstant domain, or an immune effector; expressing the proteinexpression construct in said host cell, thereby generating a recombinantfusion polypeptide; and purifying said recombinant fusion polypeptide.

In an embodiment, the spacer polynucleotide comprises a transcriptionalmodulator, a translational modifier, a constant domain, or an immuneeffector. In an embodiment, the host cell is a yeast, bacteria, ormammalian cell. In an embodiment, the method is used to generate atleast 1,000 unique recombinant fusion polypeptides. In an embodiment,the method is used to generate at least 10,000 unique recombinant fusionpolypeptides. In an embodiment, the method is used to generate at least100,000 unique recombinant fusion polypeptides.

Also provided herein is a method of enriching a mixture of unique fusionpolypeptides, comprising: providing a polynucleotide constructcomprising a first polynucleotide encoding a first variable domain, asecond polynucleotide encoding a second variable domain, and a spacerpolynucleotide linking the first and second polynucleotides; expressingthe polynucleotide construct in a population of host cells to generate apolypeptide comprising said first and second variable domain on thesurface of said host cell; enriching for a subpopulation of host cellsexpressing polypeptides that bind to said antigen of interest; andinserting a third polynucleotide into said spacer polynucleotide in oneor more said polynucleotide constructs isolated from said enrichedsubpopulation, wherein said third polynucleotide comprises atranscriptional modulator, a translational modifier, a constant domain,or an immune effector.

Also provided herein is a composition comprising a pool of at least10,000 aqueous droplets in an oil solution, wherein said aqueousdroplets have a diameter of between 1 micron and 200 microns, andwherein said aqueous droplets have an outer layer comprising asurfactant boundary, and wherein a plurality of said droplets comprise afirst probe comprising a first polynucleotide having a length of between20 and 120 nucleotides, wherein said first polynucleotide iscomplementary to a first subsequence of a polynucleotide encoding aconstant domain of an antibody or T cell receptor, and a second probecomprising a second polynucleotide having a length of between 20 and 120nucleotides, wherein said second polynucleotide is complementary to asecond subsequence of a polynucleotide encoding a constant domain of anantibody or T cell receptor; wherein said first and second probes areattached to at least one particle.

In an embodiment, the pool of at least 10,000 aqueous droplets ismonodisperse. In an embodiment, the surfactant boundary separates theaqueous phase in said droplet from the oil solution. In an embodiment,the particle is a spherical bead comprising agarose, glass, chemicalpolymers, or magnetic materials. In an embodiment, the probes comprisebiotin and wherein said particle comprises streptavidin attached to thesurface of the particle. In an embodiment, the attachment of said firstprobe or said second probe to said particle comprises a covalent bond.

In an embodiment, the surfactant boundary comprises a surfactantselected from the group consisting of: a nonionic surfactant, azwitterionic surfactant, a sulfate, a sulfonate, and a phosphate ester.In an embodiment, the nonionic surfactant is selected from the groupconsisting of: polyethylene glycol alkyl ether, sorbitan alkyl ester,and polyethylene glycol octophenol ether. In an embodiment, thesurfactant boundary comprises sodium lauryl sulfate or sodium dodecylsulfate.

Also provided herein is a composition or kit comprising a first nucleicacid probe attached to one or more beads, and a second nucleic acidprobe attached to one or more beads, wherein said first nucleic acidprobe is capable of hybridizing to a complementary region on a firsttarget polynucleotide, and wherein said second nucleic acid probe iscapable of hybridizing to a complementary region on a second targetpolynucleotide, wherein said first target polynucleotide comprises afirst variable domain, and said second target polynucleotide comprises asecond variable domain. In an embodiment, the first probe is hybridizedto said first target polynucleotide, and wherein said second probe ishybridized to said second target polynucleotide.

Also provided herein is a method for identifying antibodies of interest,comprising providing primary immune cells from at least one mammaliandonor; isolating single immune cells or subpopulations of immune cellsfrom said primary immune cells in a reaction vessel; generating aplurality of recombinant fusion polynucleotides each comprising a firstpolynucleotide encoding a light chain variable domain polypeptide and asecond polynucleotide encoding a heavy chain variable domainpolypeptide, wherein said first and second polynucleotides are each fromsaid immune cells or subpopulations of immune cells, wherein saidrecombinant fusion polynucleotides further comprise a linkerpolynucleotide linking said first and second polynucleotides; insertingat least one of each of said plurality of recombinant fusionpolynucleotides into a plurality of expression vectors; expressing saidexpression vectors in a host cell to generate a plurality of recombinantimmunoglobulins; and identifying therapeutic antibodies from saidplurality of recombinant immunoglobulins that bind to an antigen ofinterest.

In an embodiment, the at least one mammalian donor is selected for thepresence of a particular medical condition. In an embodiment, themedical condition comprises a heightened immune response to hepatitis B,rabies, tetanus toxin, varicella-zoster, cytomegalovirus, orpneumococcus. In an embodiment, the at least one mammalian donor ishuman. In an embodiment, the at least one said mammalian donor or cellsderived from said donor were exposed to an antigen of interest beforeproviding said primary immune cells. In an embodiment, the antigen ofinterest is related to a tumor or cancerous cell or tissue. In anembodiment, the antigen of interest is associated with an autoimmunedisease. In an embodiment, the at least one mammalian donor wasimmunized against at least one antigen of interest before providing saidprimary immune cells. In an embodiment, the method further comprisesinserting a promoter into each of the recombinant fusion polynucleotidesbetween said first and second polynucleotides.

Also provided herein is a method for generating an immunoglobulin or Tcell receptor polypeptide libraries, comprising: providing g a pluralityof polynucleotide constructs, each comprising a first polynucleotideencoding a first variable domain, a second polynucleotide encoding asecond variable domain, and a spacer polynucleotide linking the firstand second polynucleotides, wherein each of said linked first and secondvariable domains are from a single cell or subpopulation of cells, andwherein said plurality of polynucleotide constructs comprises linkedfirst and second variable domains from at least 1,000 unique cells orsubpopulations of cells; for each of said plurality of polynucleotideconstructs, inserting a third polynucleotide encoding a transcriptionalmodulator, a translational modifier, a constant region, or an immuneeffector into the spacer polynucleotide, thereby generating at least1,000 unique recombinant fusion polynucleotide protein expressionconstructs; performing the providing and inserting step at least 1,000times to generate at least 1,000 unique recombinant fusionpolynucleotides; performing bulk expression of said at least 1,000unique recombinant fusion polynucleotides protein expression constructsin a plurality of host cells to generate at least 1,000 uniquerecombinant fusion polypeptides, wherein said recombinant fusion proteinpolynucleotide is generated by; and purifying the at least 1,000 uniquerecombinant polypeptides, thereby generating an immunoglobulin or T cellreceptor polypeptide library.

In an embodiment, the insertion of said third polynucleotide isperformed in parallel for each of said at least 1,000 uniquepolynucleotide constructs. In an embodiment, at least one recombinantpolypeptide from said at least 1,000 unique recombinant polypeptidesbinds to Streptococcus pneumonia, Hemophilis influenza, or Klebsiellapneumonia. In an embodiment, the variable domains are amplified from asingle cell or subpopulation of cells. In an embodiment, the firstvariable domain is an immunoglobulin heavy chain, and said secondvariable domain is an immunoglobulin light chain.

In an embodiment, the first variable domain is a T cell receptor alphaand said second variable domain is a T cell receptor beta. In anembodiment, the recombinant fusion polynucleotide protein expressionconstruct is an expression vector. In an embodiment, the expressionvector is a plasmid or a phagemid. In an embodiment, the insertion ofsaid third polynucleotide into said spacer polynucleotide comprises amethod selected from the group consisting of: Gibson assembly,site-specific digestion and ligation, and targeted recombination.

Also provided herein is a composition comprising at least 1,000 uniquerecombinant fusion polynucleotide protein expression constructscomprising a first polynucleotide encoding a first variable domain, asecond polynucleotide encoding a second variable domain, and a spacerpolynucleotide linking the first and second polynucleotides, whereinsaid spacer polynucleotide comprises a transcriptional modulator, atranslational modifier, a constant region, or an immune effector.

In an embodiment, the first variable domain and said second variabledomain of each individual recombinant fusion protein expressionconstruct from said at least 1,000 unique recombinant fusion proteinexpression constructs are from a single cell or subpopulation of cells.In an embodiment, the first variable domain is an immunoglobulin heavychain, and said second variable domain is an immunoglobulin light chain.In an embodiment, the first variable domain is a T cell receptor alphaand said second variable domain is a T cell receptor beta. In anembodiment, the recombinant fusion polynucleotide expression recombinantfusion protein expression construct is an expression vector. In anembodiment, the expression vector is a plasmid or a phagemid.

Also provided herein is a method of generating a recombinantimmunoglobulin library, comprising: providing primary immune cells fromat least one mammalian donor; isolating single immune cells orsubpopulations of immune cells from said primary immune cells inreaction vessels; generating a plurality of recombinant fusionpolynucleotides each comprising a first polynucleotide encoding a heavychain variable domain polypeptide and a second polynucleotide encoding alight chain variable domain polypeptide from each cell or subpopulationof cells, wherein said recombinant fusion polynucleotides furthercomprise a linker polynucleotide linking said first and secondpolynucleotides; inserting at least one of each of said plurality ofrecombinant fusion polynucleotides into a plurality of expressionvectors; inserting a promoter into each of the recombinant fusionpolynucleotides between said first and second polynucleotides, therebygenerating a recombinant immunoglobulin encoding polynucleotide;inserting at least one of said expression vectors comprising saidrecombinant immunoglobulin encoding polynucleotide into a plurality ofhost cells; and expressing said recombinant immunoglobulin encodingpolynucleotide in each of said plurality of host cells, therebygenerating a recombinant immunoglobulin library.

In an embodiment, the at least one mammalian donor is selected for thepresence of a particular medical condition. In an embodiment, themedical condition comprises a heightened immune response to hepatitis B,rabies, tetanus toxin, varicella-zoster, cytomegalovirus, orpneumococcus. In an embodiment, the at least one mammalian donor ishuman. In an embodiment, the at least one mammalian donor or cellsderived from said donor was exposed to an antigen of interest beforeproviding said primary immune cells. In an embodiment, the antigen ofinterest is related to a tumor or cancerous cell or tissue.

In an embodiment, the antigen of interest is associated with anautoimmune disease.

In an embodiment, the at least one mammalian donor was immunized againstan antigen of interest before providing said primary immune cells. In anembodiment, the recombinant immunoglobulin library comprises at least1,000, 10,000, 100,000, or 1,000,000 unique recombinant fusionpolynucleotides. In an embodiment, the method further comprisesexpressing said recombinant immunoglobulin on the surface of apopulation of host cells, contacting said host cell population with anantigen, and selecting for a subpopulation of host cells that bind tosaid antigen to enrich said recombinant immunoglobulin library.

Disclosed herein are methods and compositions for generating recombinantfusion polypeptides. A first nucleic acid probe attached to a particleand a second nucleic acid probe attached to a particle are providedaccording to an embodiment of the invention. The first and secondnucleic acid probes are attached to the same or different particles. Theprobes are combined with an immune cell population in a reaction vessel.The first nucleic acid probe hybridizes to a first target polynucleotideform a cell or population of cells, and the second nucleic acid probehybridizes to a second target polynucleotide from the cell or populationof cells. The immune cell population is lysed and the captured targetpolynucleotides are amplified and fused, thus generating the recombinantfusion polynucleotide.

Also disclosed herein is a recombinant protein expression constructuseful for generating recombinant protein libraries. The construct isgenerated by first amplifying individual protein-coding polynucleic acidcomponents, and then fusing those components into a single polynucleicacid construct with a polynucleic acid linker. The linker is thenreplaced with polynucleic acid sequences required for efficientrecombinant expression of the proteins. This construct can be used togenerate a single recombinant protein, but can also be used to generatea pool or library of hundreds, thousands, or millions of proteins.

Also provided herein are methods of enriching a mixture of unique fusionpolypeptides.

Also provided herein are mixtures of recombinant fusion polypeptidesgenerated by the method disclosed herein. Also provided herein is acomposition comprising a pool of at least 10,000 aqueous droplets in oilsolution comprising probes and target sequences for amplification andfusion.

Also disclosed herein is a recombinant protein expression constructcomprised of a polynucleic acid, which is used for generatingrecombinant protein libraries. The construct is generated by firstamplifying individual protein-coding polynucleic acid components, andthen fusing those components into a single polynucleic acid constructwith a polynucleic acid linker. The linker is then replaced withpolynucleic acid sequences required for efficient recombinant expressionof the proteins. This construct can be used to generate a singlerecombinant protein, but can also be used to generate a pool or libraryof hundreds, thousands, or millions of proteins.

In one embodiment of the invention, nucleic acid sequences encodingantibody subunits are amplified from antibody-producing cells and thenfused into a single polynucleic acid construct with a polynucleic acidlinker. The linker is then replaced with polynucleic acid sequencesrequired for efficient recombinant expression of the proteins, such thatan antibody library can be produced en masse.

Disclosed herein are methods and compositions for generating recombinantfusion polypeptides. A first nucleic acid probe attached to a particleand a second nucleic acid probe attached to a particle are providedaccording to an embodiment of the invention. The first and secondnucleic acid probe are attached to the same or different particles. Theprobes are combined with an immune cell population in a reaction vessel.The first nucleic acid probe hybridizes to a first target polynucleotideform a cell or population of cells, and the second nucleic acid probehybridizes to a second target polynucleotide from the cell or populationof cells. The immune cell population is lysed and the captured targetpolynucleotides are amplified and fused, thus generating the recombinantfusion polynucleotide.

Industrial applications include protein therapeutics, moleculardiagnostics, and cell therapeutics. In the area of protein therapeutics,drug developers immunize a mouse with a protein target of interest, andthen extract primary B cells from that mouse. Some but not all of theseB cells produce antibodies against the target of interest. In oneembodiment, the current invention is used to extract theantibody-producing components of such B cells and then express theproteins in recombinant cells. Recombinant expression enablesprotein-screening protocols that are difficult to perform with primary Bcells. In another embodiment of the invention, the constructs are usedto produce polyclonal antibody libraries for targeted therapy orprophylaxis of particular pathogens.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis insteadplaced upon illustrating the principles of various embodiments of theinvention.

FIGS. 1A-1D show a method for generating initial linked construct usinggene-targeting probes encapsulated with nucleic acids from individualgene-expressing cells inside monodisperse droplets.

FIG. 2 shows an scFv expression construct comprising a linked heavychain and light chain variable domain encoding polynucleotide from asingle cell.

FIG. 3 shows a method for generating an immunoglobulin expressionconstruct from initial linked variable domain encoding polynucleotidesfrom a single cell.

FIG. 4 shows a method for selecting libraries of cells expressingantibody fragments with affinity for an antigen of interest.

FIG. 5 shows a Western blot showing expression of scFv antibodyfragments in yeast over time. scFvs are tagged with the peptide markerc-myc. scFvs are tagged with the peptide marker c-myc. Lanes 1-6 areinduced using the wild type AOX1 promoter at 72 hr, 66 hr, 47 hr, 24 hr,and 18 hr respectively. Lanes 7-12 are the same time points as Lanes1-6, respectively, but using a different promoter. Lane 13 is a sizemarker.

FIG. 6 shows a Western blot showing expression of full-length antibodiesin yeast. Lane 1 is a size marker. Lane 1 is a size marker. Lanes 2-5are antibodies expressed in yeast, Lane 6 is antibody expressed in CHO,all under reduced conditions. Lanes 7-10 are the same antibodiesexpressed in yeast, Lane 11 is antibody expressed in CHO, undernon-reduced conditions.

DETAILED DESCRIPTION

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

Definitions

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein, the term “recombinant” refers to a biomolecule, e.g., agene or protein, that (1) has been removed from its naturally occurringenvironment, (2) is not associated with all or a portion of apolynucleotide in which the gene is found in nature, (3) is operativelylinked to a polynucleotide which it is not linked to in nature, or (4)does not occur in nature. The term “recombinant” can be used inreference to cloned DNA isolates, chemically synthesized polynucleotideanalogs, or polynucleotide analogs that are biologically synthesized byheterologous systems, as well as proteins and/or mRNAs encoded by suchnucleic acids.

As used herein, the term “nucleic acid” refers to any materialscomprised of DNA or RNA. Nucleic acids can be made synthetically or byliving cells.

As used herein, the term “polynucleotide” refers to a polymeric chain ofnucleotides. The term includes DNA molecules (e.g., cDNA or genomic orsynthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as wellas analogs of DNA or RNA containing non-natural nucleotide analogs,non-native inter-nucleoside bonds, or both. The nucleic acid can be inany topological conformation. For instance, the nucleic acid can besingle-stranded, double-stranded, triple-stranded, quadruplexed,partially double-stranded, branched, hair-pinned, circular, or in apadlocked conformation.

Unless otherwise indicated, and as an example for all sequencesdescribed herein under the general format “SEQ ID NO:”, “nucleic acidcomprising SEQ ID NO:1” refers to a nucleic acid, at least a portion ofwhich has either (i) the sequence of SEQ ID NO:1, or (ii) a sequencecomplementary to SEQ ID NO:1. The choice between the two is dictated bythe context. For instance, if the nucleic acid is used as a probe, thechoice between the two is dictated by the requirement that the probe becomplementary to the desired target.

As used herein, the term “protein” or refers to large biologicalmolecules, or macromolecules, consisting of one or more chains of aminoacid residues. Many proteins are enzymes that catalyze biochemicalreactions and are vital to metabolism. Proteins also have structural ormechanical functions, such as actin and myosin in muscle and theproteins in the cytoskeleton, which form a system of scaffolding thatmaintains cell shape. Other proteins are important in cell signaling,immune responses, cell adhesion, and the cell cycle. However, proteinsmay be completely artificial or recombinant, i.e., not existingnaturally in a biological system.

As used herein, the term “polypeptide” refers to bothnaturally-occurring and non-naturally-occurring proteins, and fragments,mutants, derivatives and analogs thereof. A polypeptide may be monomericor polymeric. A polypeptide may comprise a number of different domainseach of which has one or more distinct activities.

As used herein, the term “antigen” refers to a biomolecule that bindsspecifically to the respective antibody. An antibody from the diverserepertoire binds a specific antigenic structure by means of its variableregion interaction (CDR loops), an analogy being the fit between a lockand a key.

As used herein, the term “antibody” (i.e., “immunoglobulin” (Ig)) refersto a polypeptide, at least a portion of which is encoded by at least oneimmunoglobulin gene, or fragment thereof, and that can bind specificallyto a desired target molecule. The term includes naturally-occurringforms, as well as fragments and derivatives. The antibody recognizes aunique part of a foreign target, called an antigen. Each tip of the “Y”of an antibody contains a paratope (a structure analogous to a lock)that is specific for one particular epitope (similarly analogous to akey) on an antigen, allowing these two structures to bind together withprecision. Using this binding mechanism, an antibody can tag a microbeor an infected cell for attack by other parts of the immune system, orcan neutralize its target directly (for example, by blocking a part of amicrobe that is essential for its invasion and survival). The antibodyconsists of four polypeptide chains; two identical “heavy chains” andtwo identical “light chains” connected by disulfide bonds. The heavychain is the longer than the light chain, and has two regions, theconstant region and the variable region. The constant region isidentical in all antibodies of the same isotype, but differs inantibodies of different isotypes. A light chain has two successivedomains: one constant domain and one variable domain. The approximatelength of a light chain is 211 to 217 amino acids. Each antibodycontains two light chains that are always identical; only one type oflight chain, κ or λ, is present per antibody in mammals.

Fragments within the scope of the term “antibody” include those producedby digestion with various proteases, those produced by chemical cleavageand/or chemical dissociation and those produced recombinantly, so longas the fragment remains capable of specific binding to a targetmolecule. Among such fragments are Fab, Fab′, Fv, F(ab′)2, and singlechain Fv (scFv) fragments.

Derivatives within the scope of the term include antibodies (orfragments thereof) that have been modified in sequence, but remaincapable of specific binding to a target molecule, including:interspecies chimeric and humanized antibodies; antibody fusions;heteromeric antibody complexes and antibody fusions, such as diabodies(bispecific antibodies), single-chain diabodies, and intrabodies (see,e.g., Intracellular Antibodies: Research and Disease Applications (1998)Marasco, ed., Springer-Verlag New York, Inc.), the disclosure of whichis incorporated herein by reference in its entirety).

Antibodies may be produced by any known technique, including harvestfrom cell culture of native B lymphocytes, harvest from culture ofhybridomas, recombinant expression systems and phage display.Recombinant antibodies may also be produced by the methods as describedherein.

As used herein, the term “B cell receptor” or “BCR” refers to atransmembrane receptor protein located on the outer surface of B-cells.The B cell receptor's binding moiety is composed of a membrane-boundantibody that, like all antibodies, has a unique and randomly determinedantigen-binding site comprising variable domains.

As used herein, the term “region” refers to a physically contiguousportion of the primary structure of a biomolecule. In the case ofproteins, a region is defined by a contiguous portion of the amino acidsequence of that protein.

As used herein, the term “domain” refers to a structure of a biomoleculethat contributes to a known or suspected function of the biomolecule.Domains may be co-extensive with regions or portions thereof; domainsmay also include distinct, non-contiguous regions of a biomolecule.Examples of protein domains include, but are not limited to, a constantdomain, a variable domain, a light chain domain, and a heavy chaindomain.

As used herein, the term “variable region” or “variable domain” refersto the antigen binding region that is highly variable. This variabilityprovides slightly different tip structures, or antigen-binding sites,allowing millions of antibodies with slightly different tip structures,or antigen-binding sites, to exist. Each of these variants can bind to adifferent antigen. This enormous diversity of antibodies allows theimmune system to recognize an equally wide variety of antigens. Thelarge and diverse population of antibodies is generated by randomcombinations of a set of gene segments that encode differentantigen-binding sites (or paratopes), followed by random mutations inthis area of the antibody gene, which create further diversity. T cellreceptor molecules also have variable regions or variable domains.

Each antibody heavy or light chain has two regions, the constant regionand the variable region. The “constant domain” is identical in allantibodies of the same isotype, but differs in antibodies of differentisotypes. Isotypes of heavy chain include IGG1, IGG2, IGE, and IGA.Different isotypes have particular “effector” functions in the immunesystem, i.e., they activate particular biological pathways for immunity.T cell receptor molecules also have constant domains.

As used herein, the term “single chain variable fragment” (scFv) refersto a single chain antibody fragment comprised of a heavy and light chainlinked by a peptide linker. In some cases scFv are expressed on thesurface of an engineered cell, for the purpose of selecting particularscFv that bind to an antigen of interest.

As used herein, the term “fusion polypeptide” or “fusion protein” refersto a polypeptide comprising a polypeptide or protein fragment coupled toheterologous amino acid sequences. Fusion proteins are useful becausethey can be constructed to contain two or more desired functionalelements from one or more proteins. A fusion protein comprises at least10 contiguous amino acids from a polypeptide of interest, at least 20 or30 amino acids, at least 40, 50 or 60 amino acids, or at least 75, 100or 125 amino acids. Fusion proteins can be produced recombinantly byconstructing a nucleic acid sequence which encodes the polypeptide or afragment thereof in frame with a nucleic acid sequence encoding adifferent protein or peptide and then expressing the fusion protein.Alternatively, a fusion protein can be produced chemically bycrosslinking the polypeptide or a fragment thereof to another protein.

As used herein, the term “transcriptional modulator” refers to anypolynucleotide sequence that modulates transcription at a location incis or trans to the transcriptional modulator. Typically, the mechanismof the transcriptional modulator is to effect binding of a transcriptionfactor protein, or complex of proteins. Examples of transcriptionalmodulators include, but are not limited to, transcriptional promoters(i.e., “promoters”) and transcriptional enhancers (i.e., “enhancers”).Transcriptional promoters are sequences at the 5′ end of genes thatmodulate expression of genes. Transcriptional enhancers arepolynucleotide sequences, typically in cis to the modulated gene, whicheffect binding of transcription factors.

As used herein, the term “translational modifier” or “translationalmodulator” refers to a polynucleotide or polypeptide sequence whichmodulates translation of a gene transcript, such as an internal ribosomeentry site (IRES).

As used herein, the term “immune effector” refers to a peptide orprotein sequence that selectively binds to another protein and therebyregulates immune activity Immune activity is regulated through anincrease or decrease in enzyme activity, gene expression, or cellsignaling, in such a way that immune cells are activated, de-activated,caused to divide, or caused not to divide. In some cases, effectormolecules are secreted, such as cytokines and antibodies. In othercases, effector molecules are affixed to the surface of a cell.

As used herein, the term “protein expression construct” refers to anypolynucleic acid sequence that can be used to express a recombinantprotein. A protein expression construct contains, at a minimum, at leastone protein-encoding region, a transcription initiation site, and apromoter sequence that modulates transcription. Many protein expressionconstructs are circular, such as a plasmid or a phagemid. Many proteinexpression constructs harbor an origin of replication (ORI) that enablesself-replication of the construct inside a host cell. Other proteinexpression constructs are linear, and do not harbor an ORI. Some proteinexpression constructs are explicitly for expression in an engineeredcell, whereas other protein expression constructs are for generatingproteins in vitro without the benefit of cellular machinery. Examples ofengineered cells include bacteria, yeast, and mammalian cells (e.g.,Chinese hamster ovary or HeLa cells).

As used herein, the term “expression vector” refers to a nucleic acidmolecule capable of introducing a protein expression construct into ahost cell. Some expression vectors also replicate inside host cells,which increases protein expression by the protein expression construct.One type of vector is a “plasmid,” which refers to a circular doublestranded DNA loop into which additional DNA segments may be ligated.Other vectors include cosmids, bacterial artificial chromosomes (BAC)and yeast artificial chromosomes (YAC), fosmids, phage and phagemids.Another type of vector is a viral vector, wherein additional DNAsegments may be ligated into the viral genome (discussed in more detailbelow). Certain vectors are capable of autonomous replication in a hostcell into which they are introduced (e.g., vectors having an origin ofreplication which functions in the host cell). Other vectors can beintegrated into the genome of a host cell upon introduction into thehost cell, and are thereby replicated along with the host genome.Moreover, certain preferred vectors are capable of directing theexpression of genes to which they are operatively linked. Such vectorsare referred to herein as “recombinant expression vectors” (or simply“expression vectors”).

As used herein, the term “polynucleotide probe” (or “nucleic acidprobe”) refers to any nucleic acid sequence that is complementary to orbinds under stringent conditions to a target nucleic acid sequence. Thepolynucleotide probe can be used to detect, capture, and/or amplify thattarget nucleic acid sequence. Polynucleotide probes include but are notlimited to DNA origami structures that include 10-5000 individualoligonucleotide components. One example of a polynucleotide probe is aprimer used in PCR amplification.

As used herein, the term “stringent conditions” refers to conditionsunder which a compound of the invention will hybridize to its targetsequence, but to a minimal number of other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances and in the context of this invention, “stringentconditions” under which oligomeric compounds hybridize to a targetsequence are determined by the nature and composition of the oligomericcompounds and the assays in which they are being investigated.

As used herein, the term “fused nucleic acid” or “recombinant fusionpolynucleotide” refers to a fusion of two nucleic acid sequences into asingle contiguous nucleic acid molecule. Fusion can be achieved throughmolecular methods that fuse nucleic acids, such as overlap extension PCRor ligation.

As used herein, the term “reaction vessel” refers to any entity thatprovides physical separation of a reaction into separate compartments.The reaction vessel may be used, for example, for screening orperforming reactions a particular cell, cell subpopulation, or nucleicacid target to the exclusion of others. Reaction vessels may becomprised of a plastic compartment, microfluidic chamber, or a droplet,e.g. of an aqueous reaction solution.

As used herein, the term “surfactant” refers to compounds that lower thesurface tension (or interfacial tension) between two liquids or betweena liquid and a solid. Surfactants are typically organic compounds thatare amphiphilic, meaning they contain both hydrophobic groups (theirtails) and hydrophilic groups (their heads). Therefore, a surfactantcontains both a water insoluble (or oil soluble) component and a watersoluble component. Surfactants will diffuse in water and adsorb atinterfaces between air and water or at the interface between oil andwater, in the case where water is mixed with oil. The water-insolublehydrophobic group may extend out of the bulk water phase, into the airor into the oil phase, while the water-soluble head group remains in thewater phase. Surfactants may act as detergents, wetting agents,emulsifiers, foaming agents, and dispersants.

As used herein, the term “aqueous” refers to a solution containingwater, typically as a solvent or medium.

As used herein, the term “oil” refers to any neutral, nonpolar chemicalsubstance that is a viscous liquid at ambient temperatures and is bothhydrophobic (immiscible with water, literally “water fearing”) andlipophilic (miscible with other oils, literally “fat loving”).

As used herein, the term “droplet” refers to a small quantity of liquid.Droplets are typically spherical, but may be comprised of cylindricalslugs that span the full diameter of a microfluidic channel Droplets mayform in air, oil, or aqueous solutions, depending on their compositionof matter and the method of formation. Droplets occur in bothmonodisperse and polydisperse populations.

As used herein, the term “monodisperse” refers to a property ofcomponents characterized by uniform or nearly uniform size. For example,monodisperse droplets typically require size dispersity <5% for >90% ofthe droplets in a mixture. In many cases, monodisperse dropletpopulations are more stable than droplet populations that are notmonodisperse, i.e., polydisperse droplet populations. In someembodiments, generation of monodisperse droplets requires some kind ofcontrolled microfluidic device.

As used herein, the term “cell” refers to the smallest unit of anorganism that can independently replicate. Cells are typicallymicroscopic and have a cytoplasm and a nucleus enclosed in a membrane,either from either a single cell organism or derived from amulticellular organism.

As used herein, the term “cell population” or “cell subpopulation”refers to at least two cells of similar kind or classification. Forexample, a cell subpopulation may be two cells separated from a cellpopulation by encapsulating the two cells in a droplet.

As used herein, the term “lysis” refers to the process of breaking thecell membrane of a cell or cells through physical or chemical means.Lysis may be achieved through a chemical surfactant such as TritonX-100, an alkaline lysis buffer, heat, electrical currents, or physicaldisruption.

As used herein, the term “DNA origami” refers to the nanoscale foldingof DNA to create arbitrary two and three dimensional shapes at thenanoscale. The specificity of the interactions between complementarybase pairs make DNA a useful construction material, through design ofits base sequences. DNA origami can be polynucleotide probes folded intoparticles.

As used herein, the term “particle” refers to any solid substance thatis added to a complex solution of biological material (i.e., a proteinor a polynucleic acid) to capture and then physically isolate a targetbiological material. Particles may be objects such as microscopic beadsmade of materials such as latex, glass, or silica, ranging in size from0.1 micron to 1 mm. Particles can also refer to nanoscale-folded DNAfrom DNA origami.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used and will beapparent to those of skill in the art. All publications and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. The materials, methods, and examples areillustrative only and not intended to be limiting.

Throughout this specification and claims, the word “comprise” orvariations such as “comprises” or “comprising”, will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Methods for Isolating Single Cells and Target Polynucleotides

In some embodiments, a single cell or alternatively subpopulation ofcells is isolated to capture DNA or RNA from each cell or subpopulationof cells. In one embodiment, the cells are from a heterogeneous pool ofT or B cells. In some embodiments, the cells are primary B cells or Tcells. In some embodiments, the cells are provided from a single person.

In some embodiments, a microfluidic device is used to generate singlecell emulsion droplets. The microfluidic device ejects single cells inaqueous reaction buffer into a hydrophobic oil mixture. The device cancreate thousands of emulsion microdroplets per minute. After theemulsion microdroplets are created, the device ejects the emulsionmixture into a trough. The mixture can be pipetted or collected into astandard reaction tube for thermocycling.

Custom microfluidics devices for single-cell analysis are routinelymanufactured in academic and commercial laboratories (Kintses et al.,2010 Current Opinion in Chemical Biology 14:548-555). For example, chipsmay be fabricated from polydimethylsiloxane (PDMS), plastic, glass, orquartz. In some embodiments, fluid moves through the chips through theaction of a pressure or syringe pump Single cells can even bemanipulated on programmable microfluidic chips using a customdielectrophoresis device (Hunt et al., 2008 Lab Chip 8:81-87). In oneembodiment, a pressure-based PDMS chip comprised of flow focusinggeometry manufactured with soft lithographic technology is used(Dolomite Microfluidics (Royston, UK)) (Anna et al., 2003 AppliedPhysics Letters 82:364-366). The stock design can typically generate10,000 aqueous-in-oil monodisperse microdroplets per second at sizeranges from 10-150 μm in diameter. In some embodiments, the hydrophobicphase will consist of fluorinated oil containing an ammonium salt ofcarboxy-perfluoropolyether, which ensures optimal conditions formolecular biology and decreases the probability of droplet coalescence(Johnston et al., 1996 Science 271:624-626). To measure periodicity ofcell and droplet flow, images are recorded at 50,000 frames per secondusing standard techniques, such as a Phantom V7 camera or Fastec InLine(Abate et al., 2009 Lab Chip 9:2628-31).

The microfluidic system can optimize microdroplet size, input celldensity, chip design, and cell loading parameters such that greater than98% of droplets contain a single cell. There are three common methodsfor achieving such statistics: (i) extreme dilution of the cellsolution; (ii) fluorescent selection of droplets containing singlecells; and (iii) The microfluidic device uses extreme cell dilution tocontrol the multi-hit rate and fluorescent cell sorting to reduce thenegative rate.

In some embodiments, input cell flow is aligned with droplet formationperiodicity, such that greater than 98% of droplets contain a singlecell (Edd et al., 2008 Lab Chip 8:1262-1264; Abate et al., 2009 Lab Chip9:2628-31). In these microfluidic devices, a high-density suspension ofcells is forced through a high aspect-ratio channel, such that the celldiameter is a large fraction of the channel's width. The chip isdesigned with a 27 μm×52 μm rectangular microchannel that flows cellsinto microdroplets at >10 μL/min (Edd et al., 2008 Lab Chip8:1262-1264). A number of input channel widths and flow rates are testedto arrive at an optimal solution.

In certain embodiments of the invention, microfluidic chips are used toisolate 10, 100, 1000, 10,000, 100,000, 1 million, or 1 billion singlecells from a heterogeneous pool of T or B cells. In some embodiments,the methods of the invention use single cells in reaction containers,rather than emulsion droplets. Examples of such reaction containersinclude 96 well plates, 0.2 mL tubes, 0.5 mL tubes, 1.5 mL tubes,384-well plates, 1536-well plates, etc.

In some embodiments of the invention, cells are encapsulated with PCRreagents. The PCR reagent mixture is specifically designed to lyse thecells, thus releasing DNA or RNA targets of interest. The PCR reagentsthen amplify a plurality of the DNA or RNA targets of interest. In someembodiments of the invention, the PCR products are linked heavy andlight chain immunoglobulin variable regions. In some embodiments of theinvention, the PCR products are polynucleotides that encode scFvpolypeptides.

In some embodiments of the invention, single cells are encapsulated intodroplets with beads comprising bound polynucleic acid probes withsequences that are complementary to polynucleic acid targets of interestin the single cells. In certain embodiments, the probes are 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, or 130 nucleotides in length. Theprobes are RNA, DNA, locked nucleic acid (LNA), peptide nucleic acid(PNA), glycol nucleic acid (GNA), or any nucleic acid analogue. Thepolynucleic acid targets are either DNA or RNA. In some embodiments ofthe invention, the polynucleic acid probes target the constant region ofT cell receptor or immunoglobulin. In some embodiments, the polynucleicacid probes target the constant region of IgK or IgG. The reagentmixture present in these embodiments comprising beads is specificallydesigned to both lyse the cells and encourage polynucleic acidhybridization, thus allowing the beads to capture DNA or RNA targets ofinterest.

In certain embodiments, polynucleic acid probes for both heavy and lightchain immunoglobulin are bound to beads. In other embodiments, probesfor heavy chain are bound to one pool of beads, and probes for lightchain are bound to a second pool of beads, and then both pools of beadsare encapsulated into droplets with single cells. In some embodiments,5′-amino-modified polynucleic acid probes are bound to carboxylic acidbeads using 2-(N-morpholino) ethane sulfonic acid (MES) buffer (Kojimaet al., 2005, Nucleic Acids Research 33:e150). In other embodiments,biotinylated polynucleic acid probes are bound to streptavidin-coatedbeads.

In some embodiments, the methods of the invention use single cells inreaction containers, rather than emulsion droplets. Examples of suchreaction containers include 96 well plates, 0.2 mL tubes, 0.5 mL tubes,1.5 mL tubes, 384-well plates, 1536-well plates, etc. A variety of otherdesigns of microfluidic chips are also used to isolate single cells(Marcus et al., 2006, Anal Chem 78:3084-3089).

In some embodiments, the cell or subpopulation of cells is added to areaction container along with beads comprising bound polynucleic acidprobes and a lysis buffer. The lysis buffer lyses the cells to allow thepolynucleic acid probes to bind to the polynucleic acid targets ofinterest from the cell or cells. The beads hybridized to the polynucleicacid targets are isolated from the lysis buffer, as single beads orsubpopulations of beads into reaction vessels, and are contacted with aPCR mix to allow amplification and or fusion of the polynucleic acidtargets.

In certain embodiments of the invention, the aqueous phase of thedroplet emulsions containing beads and their bound targets is recoveredusing a solvent such as ethyl ether. The beads are isolated intoemulsions with a PCR mix, such that, on average, single beads areisolated into single emulsion microdroplets (DeKosky et al., 2015, NatMed 21:86-91). Monodisperse emulsions can be formed on a microfluidicchip, or polydisperse emulsions can be formed using a machine such asthe IKA Utra-Turrax Tube Drive system. The PCR reagents amplify aplurality of the DNA or RNA targets of interest. In some embodiments ofthe invention, the PCR products are linked heavy and light chainimmunoglobulin variable regions. In some embodiments of the invention,the PCR products are scFv.

In certain embodiments of the invention, amplified libraries of linkedvariable region subunits are then converted into protein expressionlibraries using methods for ligation or Gibson assembly. In certainembodiments of the invention, the protein expression libraries areexpressed in a recombinant protein production system such as yeast ormammalian cells.

Methods for Amplifying and Linking Variable Regions

PCR is used to amplify many kinds of sequences, including but notlimited to SNPs, short tandem repeats (STRs), variable protein domains,methylated regions, and intergenic regions. Methods for overlapextension PCR are used to create fusion amplicon products of severalindependent genomic loci in a single tube reaction. Methods to amplifyand link variable regions are disclosed in Johnson et al., 2005 GenomeResearch 15:1315-24; U.S. Pat. No. 7,749,697; PCT Publication WO2012/083225; and PCT Publication WO 2013/096643, each of which isincorporated herein by reference in its entirety.

In some embodiments, at least two nucleic acid target sequences (e.g.,first and second nucleic acid target sequences, or first and secondloci) are chosen in the cell and designated as target loci. Forward andbackward primers are designed for each of the two nucleic acid targetsequences, and the primers are used to amplify the target sequences.“Minor” amplicons are generated by amplifying the two nucleic acidtarget sequences separately, and then fused by amplification to create afusion amplicon, also known as a “major” amplicon. In one embodiment, a“minor” amplicon is a nucleic acid sequence amplified from a targetgenomic loci, and a “major” amplicon is a fusion complex generated fromsequences amplified between multiple genomic loci, e.g., a recombinantfusion nucleotide.

The method uses “inner” primers (i.e., the reverse primer for the firstlocus and the forward primer for the second locus) comprising of onedomain that hybridizes with a minor amplicon and a second domain thathybridizes with a second minor amplicon. “Inner” primers are a limitingreagent, such that during the exponential phase of PCR, inner primersare exhausted, driving overlapping domains in the minor amplicons toanneal and create major amplicons.

PCR primers are designed against targets of interest using standardparameters, i.e., melting temperature (Tm) of approximately 55-65° C.,and with a length 20-50 nucleotides. The primers are used with standardPCR conditions, for example, 1 mM Tris-HCl pH 8.3, 5 mM potassiumchloride, 0.15 mM magnesium chloride, 0.2-2 μM primers, 200 μM dNTPs,and a thermostable DNA polymerase. Many commercial kits are available toperform PCR, such as Platinum Taq (Life Technologies), Amplitaq Gold(Life Technologies), Titanium Taq (Clontech), Phusion polymerase(Finnzymes), HotStartTaq Plus (Qiagen). Any standard thermostable DNApolymerase can be used for this step, such as Taq polymerase or theStoffel fragment.

In one embodiment, a set of nucleic acid probes (or primers) are used toamplify a first target nucleic acid sequence and a second target nucleicacid sequence to form a fusion complex (FIGS. 1A-D). As shown in FIG.1A, the first probe includes a sequence that is complementary to a firsttarget nucleic acid sequence (e.g., the 5′ end of the first targetnucleic acid sequence). The second probe includes a sequence that iscomplementary to the first target nucleic acid sequence (e.g., the 3′end of the first target nucleic acid sequence) and a second sequencethat is complementary to an exogenous sequence. In some embodiments, theexogenous sequence is a non-human nucleic acid sequence and is notcomplementary to either of the target nucleic acid sequences. Forexample, the exogenous sequence might be a polynucleotide sequence thatencodes a polypeptide sequence rich in Ser and Gly amino acids, whichlinks heavy and light chain variable regions in an scFv (see, e.g.,PCT/US1992/001478). The first and second probes are the forward primerand reverse primer for the first target nucleic acid sequence.

As shown in FIG. 1A, the third probe includes a sequence that iscomplementary to the portion of the second probe that is complementaryto the exogenous sequence and a sequence that is complementary to thesecond target nucleic acid sequence (e.g., the 5′ end of the secondtarget nucleic acid sequence). The fourth probe includes a sequence thatis complementary to the second target nucleic acid sequence (e.g., the3′ end of the second target nucleic acid sequence). The third probe andthe fourth probe are the forward and reverse primers for the secondtarget nucleic acid sequence.

The second and third probes are also called the “inner” primers of thereaction (i.e., the reverse primer for the first locus and the forwardprimer for the second locus) and are limiting in concentration, (e.g.,0.01 μM for the inner primers and 0.1 μM for all other primers). Thiswill drive amplification of the major amplicon preferentially over theminor amplicons. The first and fourth probes are called the “outer”primers.

As shown in FIGS. 1B and 1C, the first and second nucleic acid sequencesare amplified independently, such that the first nucleic acid sequenceis amplified using the first probe and the second probe, and the secondnucleic acid sequence is amplified using the third probe and the fourthprobe. Next, a fusion complex is generated by hybridizing thecomplementary sequence regions of the amplified first and second nucleicacid sequences and amplifying the hybridized sequences using the firstand fourth probes (FIG. 1D). This is called overlap extension PCRamplification.

During overlap extension PCR amplification, the complementary sequenceregions of the amplified first and second nucleic acid sequences act asprimers for extension on both strands and in each direction by DNApolymerase molecules. In subsequent PCR cycles, the outer primers primethe full fused sequence such that the fused complex is duplicated by DNApolymerase. This method produces a plurality of fusion complexes. Insome embodiments, the fusion complexes comprise a heavy chain and lightchain variable region from the same cell or subpopulation of cells. Insome embodiments, the fusion complex is used as an scFv insert.

Methods for Engineering Polynucleic Acid Protein Expression Constructs

In some embodiments, variable regions for heavy and light chainimmunoglobulin are linked and amplified, or amplified and linked, toform a plurality of polynucleic acid constructs comprising a heavy andlight chain Ig variable region connected by a linker polynucleotide, toform, e.g., an scFv encoding polynucleotide. (FIG. 2). In otherembodiments, variable regions for T cell receptor alpha and beta arelinked and amplified, or amplified and linked, to form a plurality ofpolynucleic acid constructs comprising an alpha and beta T cell receptorconnected by a linker polynucleotide. In certain embodiments, thevariable T cell receptor sequences or Ig sequences are amplified fromnaturally occurring genes, especially from single cells or clonalpopulations of cells. In other embodiments, variable T cell receptorsequences or Ig sequences are generated artificially using genesynthesis methodologies (Kosuri & Church, 2014, Nat Methods 11:499-507).In other embodiments, the library of linked complexes is generated fromcompletely artificial sequences, i.e., a large diversity (e.g., ˜10¹²unique sequences) library of randomized DNA sequences. In certainembodiments, the linked variable regions form an scFv that can beexpressed in host prokaryotic or eukaryotic cells as a recombinantsecreted or surface protein. In some embodiments, recombinant scFv areexpressed using ribosome display (Hanes et al., 1997, PNAS 94: 4937-42)or mRNA display (Mattheakis et al., 1994, Affymax Research Institute91:9022-6). In other embodiments of the current invention, linkedvariable region subunit polynucleotide constructs are never expressed asprotein, and instead serve as an intermediary polynucleotide constructor library of constructs for subsequent generation of a polynucleotideconstruct or library of polynucleotide constructs that encode afull-length protein or library of proteins. In certain embodiments ofthe invention, the libraries are comprised of one, tens, hundreds,thousands, millions, or billions of unique sequences.

In some embodiments of the invention, the polynucleotide constructcomprising the variable domain encoding polynucleotides from a singleisolated cell connected by a linker polynucleotide in a singlepolynucleotide is circularized. (see FIG. 3, top and middle panel). Forexample, if the original construct is part of a library of more than oneconstructs, then circularization is beneficial to retain any pairingbetween polynucleotide subunits amplified to form the initial linkedpolynucleotide sequence. In one embodiment, a library of scFv is createdfrom thousands of single B cells. It is often desirable to insert intothe scFv linker polynucleotide or replace at least a part of the scFvlinker polynucleotide with a promoter and other sequence elements thatare important to convert the scFv library into a library of full-lengthantibodies using DNA engineering methods described below. If the linearscFv were cleaved at the linker en masse, the resulting library would nolonger retain the native heavy and light chain variable region pairingsof the original isolated cells. Thus, it is important to circularize theconstruct before insertion of a promoter or other sequence elements intothe construct between the variable domain encoding sequences to maintainthe pairing of the original isolated cells (FIG. 3).

In some embodiments, the scFv library is circularized by first usingrestriction endonucleases to digest a plasmid and the scFv constructs,followed by ligation of the scFv constructs into the plasmid. In otherembodiments, a linear DNA construct is used instead of a plasmid.Restriction enzymes cut DNA at or near specific recognition nucleotidesequences known as restriction sites. Restriction endonucleases such asEcoRI and NotI are routinely used in molecular biology and availablecommercially from vendors such as New England Biolabs. In someembodiments of the invention, DNA ligase is then used to ligate the“sticky ends” from a plasmid with the sticky ends from the scFv library.In other embodiments of the invention, a T4 DNA ligase is used tocircularize the scFv directly, or by inserting the scFv library into alinear DNA with blunt ends. In other embodiments of the invention,Gibson assembly is used to circularize the scFv library (Gibson et al.,2008, Science 319: 1215-20). In the process of Gibson assembly, at leasttwo DNA constructs with overlapping ends are mixed in a reaction tube. A5′ exonuclease enzyme is used to chew back 5′ ends from the constructs.The constructs then anneal and a DNA polymerase is used to extend the 3′ends of the DNA. Finally, a DNA ligase seals the remaining nicks in thedouble-stranded DNA. In other embodiments of the invention, DNAengineering is achieved through homologous recombination ornon-homologous end joining.

In some embodiments of the invention, a circularized plasmid istransformed into bacteria. In some embodiments of the invention, theplasmid confers resistance to an antibiotic, and selection of thetransformed bacteria with media containing antibiotic is used togenerate a plurality of bacterial clones that contain the plasmid. Afterusing such DNA engineering methods to alter the DNA, DNA sequencing isused to verify the construct or library of constructs. DNA sequencing isperformed using massively parallel methods from vendors such asIllumina, or using single-clone sequencing methods such as Sangersequencing from vendors such as Applied Biosystems.

In certain embodiments, DNA engineering methods, such as restrictionendonucleases and Gibson assembly, as described above, are also used toinsert certain polynucleic acid sequences into the linked, circularizedpolynucleic acid construct. In certain embodiments, the insertedsequence replaces part or all of the linker polynucleotide connectingthe two variable domain encoding polynucleotide sequences. In certainembodiments of the invention, the inserted polynucleic acid is necessaryor beneficial for transcription of the recombinant construct (e.g., apromoter or enhancer) in recombinant cells (see FIG. 3, bottom panel).In other embodiments of the invention, the inserted polynucleic acidencodes protein segments important for a therapeutic modality, e g,immune effector sequences. These protein-coding segments are insertedinto the initial construct in frame, such that the resultingpolynucleotide construct produces a protein or proteins in-frame withthe original construct. In certain embodiments of the invention, theinserted components are amplified from genomic DNA or mRNA. In otherembodiments of the invention, the inserted components are synthesized invitro using DNA oligonucleotides as starting material. In certainembodiments of the invention, the scFv libraries are comprised of tens,hundreds, thousands, millions, or billions of unique sequences, and DNAengineering methods are used to insert polynucleic acid sequences intothe initial linked polynucleic acid construct en masse in a singlereaction tube. After using such DNA engineering methods to alter theDNA, DNA sequencing is used to verify the construct or library ofconstructs. DNA sequencing is performed using massively parallel methodsfrom vendors such as Illumina, or using single-clone sequencing methodssuch as Sanger sequencing from vendors such as Applied Biosystems.

In some embodiments, the method of generation of the library ofimmunoglobulins or T cell receptors is done without affinity selection.The high throughput methods described herein can be used to generate anantigen binding protein (e.g., immunoglobulin) library comprising atleast 1,000, 10,000, 100,000 or more unique antigen binding proteins,each having a variable sequences paired from a single isolated immunesell (i.e., a cognate pair). Affinity selection may also be used as anadditional step to generate a refined antigen binding protein library.

Methods for Recombinant Protein Expression

Recombinant scFv are routinely expressed in phage, in a process calledphage display (Smith, 1985, Science 228:1315-17; McCafferty et al.,1990, Nature 348:552-554). Phage display is a common laboratorytechnique for the study of protein-protein, protein-peptide, andprotein-DNA interactions that uses bacteriophages (viruses that infectbacteria) to connect proteins with the genetic information that encodesthem. Applications of phage display technology include determination ofinteraction partners of a protein (which would be used as theimmobilized phage “bait” with a DNA library consisting of all codingsequences of a cell, tissue or organism) so that the function or themechanism of the function of that protein may be determined. Phagedisplay is also a widely used method for in vitro protein evolution(also called protein engineering). As such, phage display is a usefultool in drug discovery. It is used for finding new ligands (enzymeinhibitors, receptor agonists and antagonists) to target proteins(Lunder et al., 2005, J Lipid Research 46:1512-1516; Bratkovic et al.,2005, Biochem Biophys Res Commun 332:897-903). The technique is alsoused to determine tumour antigens (for use in diagnosis and therapeutictargeting) and in searching for protein-DNA interactions usingspecially-constructed DNA libraries with randomized segments (Hufton etal., 1999, J. Immunol Methods 231: 39-51; Gommans et al., 2005, J MolBiol 354:507-519). Libraries of engineered phage are comprised ofhundreds, thousands, millions, or billions of unique scFv sequences.Large library diversity enables screening for scFv with affinity to anantigen of interest in a massively parallel fashion.

Recombinant scFv are routinely expressed on the surface of eukaryoticcells such as mammalian cells and yeast. The advantage of cell surfacedisplay is the use of quantitative flow cytometric sorting and analysisto identify high-affinity interactions and normalize for antibodyprotein expression. Minimally, polynucleotide expression constructs forscFv surface expression include polynucleotide sequences for atranscriptional promoter, a heavy chain variable region sequence, apolypeptide linker sequence, and a light chain variable region sequence.Yeast display is similar to phage display in that a recombinant scFv isengineered into a polynucleotide expression construct and thentrafficked to the surface of the yeast, using a peptide traffickingsignal such as Aga2 (Boder & Wittrup, 1997, Nat Biotech, 15:553-57).Commonly used yeast strains for recombinant protein expression includePichia pastoris and Saccharomyces cerevisiae. In some embodiments, yeastdisplay is used for the study of protein-protein, protein-peptide, andprotein-DNA interactions. Libraries of engineered yeast are comprised ofhundreds, thousands, millions, or billions of unique scFv sequences.Large library diversity enables screening for scFv with affinity to anantigen of interest in a massively parallel fashion (Dangaj et al.,2013, Cancer Res 73:4820-4829). In other embodiments, mammalian cellsare used for surface expression of scFv instead of yeast (Ho et al.,2006, PNAS 103:25). In certain embodiments, mammalian cell surfaceexpression occurs by fusing the scFv to CCR5 protein, or theplatelet-derived growth factor (PDGF) (Urban et al., 2005, Nucleic AcidsResearch 33:e35; Wolkowicz et al., 2005, J Biol Chem 280:15195-15201)Mammalian cells for recombinant protein expression include Chinesehamster ovary (CHO) cells (Anderson et al., 2004, Curr Opin Biotechnol15:456-462). Recombinant DNA is introduced into the mammalian cellgenome using a retrovirus, or introduced transiently into the mammaliancells using a self-replicating plasmid.

In certain embodiments of the sequence, polynucleotide complexescomprised of linked heavy and light chain Ig (e.g., scFv) are convertedto full-length antibody proteins for downstream applications, such asantibody therapeutics. Such applications require additional portions ofthe antibody sequences that are not present in scFv, and which would bedifficult to amplify from single cells. Additionally, in certainembodiments, because antibodies are comprised of protein product fromtwo genes (heavy and light chain Ig), a full-length antibody expressionconstruct requires two promoters, i.e., one promoter each for heavy andlight chain (see, e.g., FIG. 3, bottom panel). In other embodiments, thescFv linker is replaced with an internal ribosome entry site (IRES),which enables separate expression of heavy and light chain Ig. Cell-freesystems like ribosome display can produce large (˜10′³ to 10′⁴ uniqueantibodies) diversity libraries of antibodies (Hanes & Pluckthun, 1997,PNAS 94:4937-4942). Though cell-free systems are useful for manyapplications, problems with protein folding, posttranslationalmodifications, and codon usage limit the utility of such methods forproducing fully functional therapeutic antibodies. In certainembodiments of the invention, polynucleotide complexes comprised oflinked heavy and light chain Ig are converted into full-lengthexpression constructs using the methods described above. In otherembodiments of the invention, polynucleotide complexes comprised oflinked T cell receptor subunits are converted into full-lengthexpression constructs using the methods above. In certain embodiments ofthe invention, the full-length expression constructs are used to inducerecombinant protein expression in cells such as bacteria, yeast, ormammalian cells. In certain embodiments, the library of full-lengthexpression constructs is comprised of tens, hundreds, thousands,millions, or billions of different proteins. In certain embodiments ofthe invention, the full library of full-length constructs is introducedinto recombinant protein-producing cells en masse, to produce a celllibrary comprised of tens, hundreds, thousands, millions, or billions ofdifferent clones that can be used to produce a protein library comprisedof tens, hundreds, thousands, millions, or billions of differentproteins.

Protocols for full-length antibody expression in mammalian cells arewell understood, with the first commercial monoclonal antibodiesproduced in CHO reaching the market in 1997 (Rituxan). Minimally, afull-length antibody polynucleotide expression construct is comprised ofpolynucleotide sequences encoding a full-length heavy chain protein, apromoter for the heavy chain, a full-length light chain protein, and apromoter for the light chain Promoters for antibody expression includehuman cytomegalovirus (hCMV) Mammalian cells are transfected with theantibody expression constructs using methods such as electroporation,calcium phosphate precipitation, lipofection, and retroviraltransfection. These methods are used to introduce a library offull-length constructs into recombinant protein-producing cells enmasse, to produce a cell library comprised of tens, hundreds, thousands,millions, or billions of different clones that can be used to produce aprotein library comprised of tens, hundreds, thousands, millions, orbillions of different proteins. The expression vectors may includeresistance markers against compounds such as hygromycin, which can beused to select against cells that have not been successfully transfectedwith the recombinant expression vector. CHO cultures are typicallymaintained in shake flasks at 37° C. in 8% CO₂, using media availablecommercially from suppliers such as GIBCO and HyClone. In certainembodiments, the host cells are deficient in metabolic enzymes such asdihydrofolate reductase (DHFR). Cells that are deficient in DHFR areproline-required auxotrophs, so transfecting DHFR-deficient CHO cellswith vectors that contain DHFR and then growing the transfected cells inproline-deficient medium can help select clones that express thefull-length antibodies. Thus, both negative selection (e.g., hygromycin)and positive selection (e.g., rescued DHFR deficiency) can be used togenerate large libraries of mammalian cell clones that expressfull-length antibodies. In other embodiments, stable transfectants aregenerated using site-specific recombination, for example, using theCre/loxP engineering system (Kameyama et al., 2010, Biotechnol Bioeng105:1106-1114; Wiberg et al., 2006, Biotech Bioeng 94:396-405). Suchmethods enable more predictable protein expression levels across largelibraries of mammalian cell clones. In other embodiments, an artificialchromosome expression (ACE) system is used to express recombinantproteins. The ACE system consists of a mammalian-based artificialchromosome known as Platform ACE, an ACE targeting vector (ATV) and amutant integrase (ACE integrase) for targeted recombination (Kennard etal., 2009, Biotechnol Bioeng 104:540-553). Platform ACE consists ofmainly tandem repeated ribosomal genes and repetitive satellitesequences, which form the pericentromeric heterochromatin. It also hasnatural centromeres and telomeres to enable DNA replication without theneed of integration into host cell genome, reducing the probability ofchromosomal aberration and clonal heterogeneity.

In other embodiments of the invention, yeast are used to producerecombinant protein. Yeast strains commonly used for recombinant proteinproduction include Pichia pastoris and Saccharomyces cerevisiae. Incertain embodiments, production of proteins requires post-translationalprotein modifications that do not occur naturally in wild type yeast. Insuch cases, it is useful to use glyco-engineered yeast, for example, theGlycoSwitch technology, which is a family of Pichia strains that areengineered to have post-translational glycosylation that is more“humanlike”, for example, as Gal(2)GlcNAc(2)Man(3)GlcNAc(2)N-glycans(Jacobs et al., 2009, Nat Protoc, 4:58-70). Yeast are routinely culturedin media such as YPD (1% yeast extract, 2% peptone, 2% dextrose),typically at 30° C. Many different vectors are commercially available,such as the pPICZ series of vectors from Life Technologies. Typicallythese vectors contain resistance to a chemical that can be used tonegatively select un-transformed yeast, such as zeocin or kanomycin.Polynucleotide constructs or libraries of polynucleotide constructs areroutinely introduced into yeast cells using an electroporator, thoughspheroplast generation, LiCl, and polyethylene glycol methods are alsoused. In certain embodiments, the AOX1 promoter is used to induceprotein expression (Cregg et al., 2011, Methods in Enzymology463:169-187). Secretion of recombinant protein is routinely directedusing peptide signals such as alpha-MF at the NH₂ terminus of therecombinant protein. In many embodiments, it is possible to engineeryeast to generate and secrete recombinant protein at production levelsas high as 10 g/L.

In certain embodiments of the invention, it is desirable to express theproteins in primary T cells, particularly for chimeric antigen receptormodified T cells (CARs). In some embodiments of the invention, theprotein expression construct is subjected to in vitro transcription toproduce an mRNA. These mRNA are then introduced into primary T cellsusing electroporation. In other embodiments of the invention, theprotein expression constructs are retroviruses, which are transfectedinto primary T cells, incorporating the protein expression constructinto the genome.

Monoclonal Antibody Drug Discovery

Antibody therapeutics are increasingly used by pharmaceutical companiesto treat intractable diseases such as cancer (Carter 2006 Nature ReviewsImmunology 6:343-357). However, the process of antibody drug discoveryis expensive and tedious, requiring the identification of an antigen,and then the isolation and production of monoclonal antibodies withactivity against the antigen. Individuals that have been exposed todisease produce antibodies against antigens associated with thatdisease, so it is possible mine patient immune repertoires forantibodies that could be used for pharmaceutical development. However, afunctional monoclonal antibody requires both heavy and light chainimmunoglobulins.

Certain embodiments of the invention require a large library of linkedpolynucleotide constructs comprised of variable regions from heavy andlight chain Ig. In certain embodiments, the library is generated fromhundreds, thousands, millions, or billions of single B cells. B cellisolation may be performed using droplet microfluidics, or isolationinto physical containers such as 96-well plates. The library is eitherpre-enriched for a particular target of interest, or enriched throughaffinity screening as surface-expressed scFv (FIG. 4). The enrichedlibrary is then converted to a library of full-length antibodies byfirst engineering the linked polynucleotide constructs intopolynucleotide constructs that encode full-length antibodies, and thenscreening the full-length antibodies for affinity or activity against aparticular antigen. Conversion to full-length antibodies is particularlyuseful, because many scFv with affinity against an antigen do not haveaffinity against antigens when converted to full-length antibodies. Insome embodiments, the scFv DNA library is generated from primary B cellsisolated from human donors. In certain embodiments, enrichment occurs byexposing human donors to a vaccine comprised of antigens from aparticular pathogen, such that B cells from the human donors areenriched for antibodies against those antigens. In other embodiments, Bcells are isolated from donors with a particular clinical disease, suchas autoimmune disease or cancer. In other embodiments, the library oflinked Ig complexes is generated from completely artificial sequences,i.e., a large 10¹² diversity library of randomized DNA sequences.

In some embodiments of the invention, mice are immunized with a proteinor other kind of antigen of interest. Single B cells are isolated andlinked complexes of paired heavy and light chain variable regions areproduced in vitro. B cell isolation may be performed using dropletmicrofluidics, or isolation into physical containers such as 96-wellplates. In some embodiments, these libraries are expressed on thesurface of engineered cells as scFv, and then the engineered cells aresorted for binding to sequence variants of the antigen of interest. Insome embodiments, RNA or DNA is extracted from the sorted engineeredcells, and the RNA or DNA is sequenced to determine the immunoglobulincontent of the selected cells. In some embodiments, linked Ig complexesamplified from the antigen-selected engineered cells are then cloned enmasse into a circular DNA construct, such as a plasmid vector, toproduce a library of hundreds, thousands, or millions of circularizedlinked complexes. In certain embodiments, this selected library ofplasmid vectors is enriched for Ig complexes with affinity toward anantigen of interest.

To study the function of these Ig complexes as full-length antibodies,the linker sequence between the heavy and light chain variable regionpolynucleotide sequences is then replaced with a polynucleotide sequencethat encodes immunoglobulin protein subunits required for expression ofthe full length antibody. In some embodiments of this invention, theinserted polynucleotide sequence also includes a transcriptionalpromoter that drives expression of one of the Ig chains. In someembodiments of the invention, the library of protein expressionconstructs is then introduced into a population of host cells to producea library of engineered cells that express a library of hundreds,thousands, or millions of recombinant proteins. The library offull-length antibodies is then analyzed to discover monoclonalantibodies that may be of use therapeutically. In certain embodiments,functional monoclonal antibodies are discovered by first isolatingsubpopulations of engineered cells and then screening pools for affinityagainst a particular antigen. Pools of engineered that show activityagainst an antigen are then divided into single cells, and screenedagain for affinity against a single antigen. Cell isolation may beperformed using droplet microfluidics, or isolation into physicalcontainers such as 96-well plates. In this way, monoclonal antibodieswith affinity for antigens of interest are discovered.

Polyclonal Antibody Therapeutics

Intravenous immunoglobulin (IVIg) is a pool of proteins isolated fromthe plasma of thousands of donors. The US Food and Drug Administration(FDA) has approved IVIg therapy for six indications, includingidiopathic (immune) thrombocytopenic purpura (ITP), Kawasaki'svasculitis, B cell chronic lymphocytic leukemia (CLL), and primaryimmunodeficiencies (Orange et al., 2006). Though the mechanism forautoimmune modulation is unknown, most IVIg is used as replacementtherapy for patients who are deficient in antibodies (Hartung et al.,2009). IVIg sales are $7 billion worldwide and growing at 8-10% peryear, due to an aging population and ever-expanding off-label modalities(Taylor & Shapiro, 2013).

Current methods for IVIg production threaten continued expansion of IVIgtherapy because of supply chain risk, impurities, and batch-to-batchvariability. IVIg production is highly dependent on limited human serasupply and requires investment in expensive, large-scale purificationfacilities. More than 90% of global supply is in the hands of only 3companies. In 2006, demand for IVIg exceeded supply by 4%, which causedphysicians to ration supply by turning away patients and administeringlower doses (McGinnity, 2007). Because IVIg is purified from primarysera, protein impurities and the spectre of viral contamination are acontinuing problem. Octapharma recently suffered a massive voluntaryrecall of its IVIg product (Octagam 5%) because of complicationsresulting from contamination by coagulation factor XIa (Roemisch et al.,2011). IVIg depends on antigen binding through its polyclonal variableregion milieu. However, because of the vast diversity of immunerepertoires in donor populations, preps always have different variableregion content. A survey of anti-HAV antibody titers of IVIg preps from30 different pools of >60,000 donors showed high variability with a CVof 33% among pools (Simon & Spath, 2003).

In one embodiment of the invention, IVIg is produced in recombinantcells rather than extracted from donor plasma. In this embodiment,primary B cells are collected from thousands of human donors. Singlecells are isolated and linked complexes of paired heavy and light chainvariable regions are produced in vitro. B cell isolation may beperformed using droplet microfluidics, or isolation into physicalcontainers such as 96-well plates. In some embodiments, the linkedcomplexes are cloned en masse into a circular DNA construct, such as aplasmid vector, to produce a library of hundreds, thousands, or millionsof circularized linked complexes. The linker sequence between the heavyand light chain variable region polynucleotide sequences is thenreplaced with a linker construct that includes a transcriptionalpromoter and any required portions of heavy or light chain Ig. In someembodiments of the invention, the library of protein expressionconstructs is then introduced into a population of cells, to produce alibrary of engineered cells that express a library of hundreds,thousands, or millions of antibody proteins. In some embodiments, theseantibody proteins are substantially equivalent to the antibodiesproduced by the original primary B cells. The pool of antibody proteinsis therefore used as a recombinant replacement for IVIg. In someembodiments, massively parallel DNA sequencing is used to determine thediversity of the B cells, the initial library of paired heavy and lightchain Ig, the library of protein expression constructs, and/or theengineered host cells. DNA sequencing may be useful as a qualitycontrol/quality assurance step for cell banking and protein libraryproduction.

In another embodiment of the invention, patients are selected for thepresence of a particular medical condition, such as exposure to aparticular pathogen. These patients act as donors for B cells that areenriched for production of antibodies against that particular pathogen.Conventional IVIg producers already market conventional IVIghyperimmunes with heightened activity against pathogens such ashepatitis B, rabies, tetanus toxin, varicella-zoster, andcytomegalovirus (CMV). In one embodiment of the invention, B cell donorsare injected with a vaccine against pneumococcus. B cells are extractedfrom these donors and then large libraries of polynucleotide proteinexpression constructs are made from hundreds, thousands, or millions ofsingle B cells. B cell isolation may be performed using dropletmicrofluidics, or isolation into physical containers such as 96-wellplates. In certain embodiments, these protein expression constructs areintroduced into engineered cells en masse and then protein libraries areproduced from the engineered cells. These protein libraries may containhundreds, thousands, or millions of individual antibodies, depending onthe diversity of the starting input B cells. In some embodiments, theresulting protein libraries are used as targeted polyclonal therapeuticsagainst particular pathogens, such as CMV. In other embodiments of theinvention, cells are engineered to express surface scFv using a libraryof linked sequences generated from primary B cells. The scFv-expressingcells are then exposed to an antigen of interest, such as CMV antigen,to positively select for scFv with affinity for said antigen. Thelibrary of enriched cells is then used to generate a library offull-length therapeutic antibodies as described above. In someembodiments, massively parallel DNA sequencing is used to determine thediversity of the B cells, the initial library of paired heavy and lightchain Ig, the library of protein expression constructs, and/or theengineered host cells. DNA sequencing may be useful as a qualitycontrol/quality assurance step for cell banking and protein libraryproduction.

Equivalents And Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed.(Plenum Press) Vols A and B (1992).

Example 1 scFv Library Generation

Methods and compositions of the invention will now be discussed relativeto scFv library generation.

Preparation of Beads

2×LiCl buffer was prepared as follows: For 250 mL of 2×LiCl buffer, 10mL of 1M Tris (pH 7.5), 31.25 mL 8M LiCl, and 1 mL of 500 mM EDTA wereadded to 180.25 mL molecular grade water. 2× lysis buffer was preparedas follows: For each 1 mL of 2× lysis buffer, 890 μL of 2×LiCl stock, 90μL of molecular grade water, 10 ul of 1M DTT, and 10 μL of Tween 20 weremixed to produce 1 mL solution of 2× lysis buffer.

Biotinylated IgK and IgG probes, which bind to the IgK and IgG constantregions, respectively, were synthesized with the following sequences:SEQ ID NO: 6-7. Probes were each added to 100 μL of 2× lysis buffer to afinal concentration of 10 μM.

A 1 μM solution of streptavidin magnetic beads from New England Biolabs(NEB S1420S) was gently rocked at room temperature for 30 minutes. 200μL of the streptavidin magnetic bead solution was placed into a 1.5 mLtube. A strong magnet was used to remove supernatant from the beads. Thebeads were then washed with 600 μL of 2× lysis buffer, followed byremoval of supernatant from the beads using a strong magnet. Beads werethen re-suspended in 60 μL of the 10 μM IgK and IgG probes. Thebead-probe mixture was incubated at room temperature for 10 minutes.After incubation, the mixture was exposed to a magnet to remove thesupernatant. The beads were then twice washed with 200 μL of 2× lysisbuffer, followed by removal of the supernatant with a magnet after eachwash. After washing, the beads were re-suspended in 500 μL of 2× lysisbuffer (1:2.5 dilution). 5 μL of an RNase inhibitor were added to thebeads (1% final RNase concentration) and the solution was gently mixed.

Capture of RNA Transcripts from Cells on Beads in Emulsions

A microfluidic system with three pressure pumps (Dolomite microfluidics)was set up and connected to a pressure source of at least 6000 mbar. Onepressure pump chamber was filled with a solution of mineral oilcomprising 4.5% Span-80, 0.4% Tween 80, and 0.05% Triton X-100. Thesecond pressure chamber was filled with Dulbecco's Phosphate-BufferedSaline (DPBS). The third pressure pump chamber was filled with water.Each of the three pressure pumps was connected to a microfluidic dropletchip (Dolomite microfluidics) comprising inputs for oil and aqueousphases, a flow-focusing junction for droplet generation, and channelscoated with a hydrophobic material. Primary B cells were purified usinga pan-B negative selection kit (Stem Cells Inc.) and mixed with aAdalumimab-expressing Chinese hamster ovary (CHO) cell clone at 0.1%prevalence in DPBS, and then loaded into the second pressure chamber andthe bead mixture described above was loaded into the third pressurechamber. The CHO line acts as a positive control, expressing thepreviously published monoclonal antibody sequence Adalumimab(http://www.drugbank.ca/drugs/DB00051). All three pumps were theninitialized at around 50% maximum pressure. Droplet formation wasnormalized, and emulsions containing droplets with the bead/cell mixturewere collected into PCR tubes. The tubes were then incubated at 50° C.for 30 minutes. After incubation, ethyl acetate extraction wasperformed. A 2:1 volume of ethyl acetate was added to each tube,transferred to a 1.5 mL tube, followed by vortexing and centrifugationat full speed for 1 minute. After centrifugation, the supernatant wasremoved. The process was repeated with a 1:1 volume of ethyl acetateuntil enough of the emulsion had broken. After ethyl acetate extraction,the tube was placed on a strong magnet for 1-2 minutes and allsupernatant was removed and discarded.

Amplification of Linked Complexes from Beads in Emulsions to Form scFvEncoding Polynucleotide

Beads comprising probes attached to RNA transcripts collected above werethen exposed to overlap extension RT-PCR to amplify the RNA transcripts.

25 μL of the beads with bound RNA transcripts were transferred to a 1.5mL tube, and put on a magnet, allowing removal of the supernatant. Thebeads were then washed with 100 μL of water, followed by removal of thesupernatant. Next, single beads or subpopulations of beads were isolatedinto reaction chambers to amplify RNA from a single cell orsubpopulation of cells. To accomplish this, the beads were re-suspendedin 25 μL of cold RT-PCR mix (kits and enzymes from NEB, Thermo Fisher,and Qiagen). The RT-PCR mix consisted a final concentration of: lxreaction buffer, 1 μM outer IgK V primer (SEQ ID NO: 8), 0.2 μM innerIgK C primer (SEQ ID NO: 9), 0.2 μM inner IgG V primer (SEQ ID NO: 10),1 μM outer IgG C primer (SEQ ID NO: 11), 4 ng/μL ET SSB, 2% RNaseinhibitor, and 4% Reverse transcriptase and thermostable polymerase. Anemulsion comprising the beads and RT-PCR mix was formed using anemulsion generating device (IKKA ULTRA-TURRAX Tube Drive systems withDT-20 tubes). A DT-20 tube was placed on the emulsion-generating deviceand 75 μL of an oil mix comprising 4.5% Span-80, 0.4% Tween 80, 0.05%Triton X-100 in mineral oil was added to the tube. 25 μL of cold beadsand RT-PCR mix was then added dropwise to the top of the oil layer inthe DT-20 tube. Emulsions of the solution in the DT-20 tube were thenformed using the emulsion-generating device. After making emulsions, theemulsion mix was divided into 100 μL aliquots into PCR tubes.

RT-PCR was then performed on the emulsion mix using the followingthermocycle conditions sequentially:

55° C. 30 min 94° C. 3 min 94° C. 30 s 17 cycles; 65° C. to 57° C. 3 min−0.5° C. anneal per cycle 68° C. 1 min 94° C. 30 s 26 cycles 57° C. 3min [43 cycles total] 68° C. 1 min 68° C. 2 min  4° C. ∞

After PCR, 100 μL of ethyl acetate was added to each tube and thesolution was mixed with a pipette. The broken emulsions were thentransferred to a 1.5 mL tube and pulse vortexed to mix. The solution wasthen centrifuged at full speed for 1 minute and the upper layer(supernatant) was removed. The tube was then placed on a magnet and thelower layer (aqueous phase) comprising the amplification product wastransferred to new 2 mL tube without beads. A QIAquick PCR PurificationKit from Qiagen was then used to extract amplified DNA from the aqueousphase. The isolated amplified DNA includes a library of scFv inserts(scFv encoding polynucleotide) (i.e., SEQ ID NO: 13, from the controlAdalumimab sequence) that will be used in the generation of expressionconstructs.

Generation of Expression Constructs

pPIC9 vector (Life Technologies) was modified to include a portion ofthe human IgG1 sequence (SEQ ID NO: 12). The concentration of scFvinserts generated above was determined. 0.02 pmol of pPIC9_JgG1 vectorwas combined with 0.04 pmol of isolated scFv inserts (e.g., example SEQID NO: 13) in water to a final volume of 5 μL. 5 μL of 2× GibsonAssembly Master Mix (New England Biolabs) was added to the vector/scFvinsert solution. The samples were then incubated at 50° C. for 60minutes to generate a circularized construct of the scFv insert in thepPIC9_IgG1 vector.

A wild type AOX1 promoter was then added to the circularized constructto induce expression of the scFv insert as follows: 0.02 pmol of thecircularized construct comprising the scFv insert and the pPIC9_JgG1vector was combined in water to a final volume of 5 μL. 5 μL of 2×Gibson Assembly Master Mix (New England Biolabs) was added to thevector/scFv insert solution. The samples were then incubated at 50° C.for 60 minutes to generate a circularized construct of the scFv insertin the pPIC9_IgG1 vector.

The circularized construct was then linearized with XhoI restrictionendonuclease from NEB to generate linearized scFv plasmid DNA.

Protein Expression in Yeast

Frozen competent yeast cells were thawed on ice. 40 μL of yeast cellswere transferred to a tube containing 10 μL of 0.1 μg/μL linearized scFvplasmid DNA. The DNA and yeast cell mixture was incubated on ice for 5minutes. The mixture was then electroporated at 1.5 kV, 200 Omega, and25 uF. 1 mL of recovery medium (50% 1M sorbitol, 50% yeast extractpeptone dextrose (YPD)) was then added to the mixture, and the tube wasshaken at 200 rpm for 1 hour at 30° C. The yeast was then plated ontoRDB minus His agar selection plates and incubated overnight at 30° C. Atransformed yeast colony was then selected form the plate and added to50 mL BMGY medium. The yeast was shaken at 200 rpm overnight (at least20 hours) at 30° C., until reaching an OD600 of between 5 and 10. Then,the culture was centrifuged at 2000×g for 5 minutes, and the supernatantwas removed. Yeast cells were then resuspended in 50 mL BMMY medium andgrown for 24 hours at 30° C. while being shaken at 200 rpm. After 24hours, methanol was added to the yeast culture to a final concentrationof 1% volume by volume. The yeast culture was then centrifuged at 2000×gfor 5 minutes to collect the scFv library in the supernatant. Thesupernatant was collected, filtered, and stored at 4° C. for furtherpurification and analysis.

A portion of the supernatant collected from yeast was run on a Westernblot as shown in FIG. 5 to show the scFv library. scFvs are tagged withthe peptide marker c-myc. scFvs are tagged with the peptide markerc-myc. Lanes 1-6 are induced using the wild type AOX1 promoter at 72 hr,66 hr, 47 hr, 24 hr, and 18 hr respectively. Lanes 7-12 are the sametime points as Lanes 1-6, respectively, but using a different promoter.Lane 14 is a size marker.

Example 2 Antibody Generation

Generation of Expression Constructs

An scFv library (including, e.g., SEQ ID NO: 1) was linearized by PCRusing standard PCR methods and forward and reverse primers (e.g., SEQ IDNO: 4,5). An insert containing a second AOX1 promoter was synthesized bya gene synthesis vendor (IDT). A insert was then added to thecircularized construct to induce expression of the full length antibody,such that the vector would now contain two AOX1 promoters, i.e., oneeach for heavy and light chain immunoglobulin. The new library wasengineered as follows: 0.02 pmol of the linearized construct comprisingthe pPIC9_JgG1_scFv vector was combined with 0.04 pmol of the promoterinsert (SEQ ID NO: 3) in water to a final volume of 5 μL. 5 μL of 2×Gibson Assembly Master Mix (New England Biolabs) was added to the scFvvector/promoter solution. The samples were then incubated at 50° C. for60 minutes to generate a circularized construct of the promoter insertin the pPIC9_IgG1_scFv vector.

Protein Expression in Yeast

Frozen competent yeast cells were thawed on ice. 40 μL of yeast cellswere transferred to a tube containing 10 μL of 0.1 μg/μL linearized fulllength antibody DNA from above. The DNA and yeast cell mixture wasincubated on ice for 5 minutes. The mixture was then electroporated at1.5 kV, 200 Omega, and 25 uF. 1 mL of recovery medium (50% 1M sorbitol,50% YPD) was then added to the mixture, and the tube was shaken at 200rpm for 1 hour at 30° C. The yeast was then plated onto RDB minus Hisagar selection plates and incubated. A transformed yeast colony was thenselected form the plate and added to 50 mL BMGY medium. The yeast wasshaken at 200 rpm overnight (at least 20 hours) at 30° C., untilreaching an OD600 of between 5 and 10. Then, the culture was centrifugedat 2000×g for 5 minutes, and the supernatant was removed. Yeast cellswere then resuspended in 50 mL BMMY medium and grown for 24 hours at 30°C. while being shaken at 200 rpm. After 24 hours, methanol was added tothe yeast culture to a final concentration of 1% weight by volume. Theyeast culture was then centrifuged at 2000×g for 5 minutes to collectthe recombinant antibody library in the supernatant. The supernatant wascollected, filtered, and stored at 4° C. for further purification andanalysis.

A portion of the supernatant collected from yeast was run on a Westernblot as show the recombinant antibody library. FIG. 6 shows theresulting Western blot, showing expression of full-length antibodies inyeast. Lane 1 is a size marker. Lanes 2-5 are antibodies expressed inyeast, Lane 6 is antibody expressed in CHO cells, all under reducedconditions. Lanes 7-10 are the same antibodies expressed in yeast, Lane11 is antibody expressed in CHO cells, under non-reduced conditions.

Example 3 Monoclonal Antibody Drug Discovery

Mice are immunized with a protein or other kind of antigen of interest.Single B cells from the mice are isolated and linked complexes of pairedheavy and light chain variable regions are produced in vitro. B cellisolation is performed using droplet microfluidics or isolation intophysical containers such as 96-well plates. The fusion constructlibraries are inserted into and expressed in host cells on the surfaceas scFv. The engineered cells are screened for binding to sequencevariants of the antigen of interest. RNA or DNA is extracted fromengineered cells with binding affinity for the antigen of interest, andthe RNA or DNA is sequenced to determine the immunoglobulin content ofthe selected cells. The linked Ig complexes amplified from theantigen-selected engineered cells are cloned en masse into plasmidvectors to produce a library of plasmid vectors comprising therecombinant fusion construct.

To study the function of these Ig complexes as full-length antibodies,the linker sequence between the heavy and light chain variable regionpolynucleotide sequences is replaced with a polynucleotide sequence thatencodes immunoglobulin protein subunits required for expression of afull length antibody comprising the paired heavy and light chainvariable regions produced in vitro. The inserted polynucleotide sequencealso includes a transcriptional promoter that drives expression of oneof the Ig chains. The library of protein expression constructs isintroduced into a population of host cells to produce a library ofengineered cells that express a library of recombinant full-lengthantibodies. The library of recombinant full-length antibodies isanalyzed to discover monoclonal antibodies that are of usetherapeutically. To perform this analysis, the subpopulations ofengineered cells is isolated and isolated pools are screened foraffinity against a particular antigen. Pools of engineered cells thatshow activity against an antigen are divided into single cells, andscreened again for affinity against a single antigen. Cell isolation isperformed using droplet microfluidics or isolation into physicalcontainers such as 96-well plates. Recombinant antibodies with affinityfor antigens of interest are discovered.

Example 4 Polyclonal Antibody Therapeutics

In this example, IVIg is produced in recombinant cells rather thanextracted from donor plasma. Primary B cells is collected from thousandsof human donors. Single cells from each donor are isolated and linkedcomplexes of paired heavy and light chain variable regions from eachcell are produced in vitro. B cell isolation is performed using dropletmicrofluidics or isolation into physical containers such as 96-wellplates. The resulting fused protein complexes is cloned en masse intoplasmid vectors to produce a library of fused protein encoding plasmidvectors. The linker sequence between the heavy and light chain variableregion polynucleotide sequences is replaced with a linker construct thatincludes a transcriptional promoter and any required portions of heavyor light chain Ig, i.e., part of a constant region that was not includedin the original scFv expression construct. The library of fused proteinexpression constructs is introduced into a population of cells toproduce a library of engineered cells that express a library ofrecombinant antibody proteins. The recombinant antibody proteinscomprises variable light and heavy immunoglobulin domains from thesource primary B cells. The pool of antibody proteins can be used as arecombinant replacement for IVIg.

The diversity of the B cells, the initial library of paired heavy andlight chain Ig, the library of protein expression constructs, and/or theengineered host cells will be determined using massively parallel DNAsequencing.

Example 5 Polyclonal Antibody Pneumococcus Therapeutics

B cell donors are injected with a vaccine against pneumococcus. B cellsare isolated from these donors. Then, large libraries of polynucleotideprotein expression constructs are made from each of the individual Bcells. B cell isolation is performed using droplet microfluidics, orisolation into physical containers such as 96-well plates. The proteinexpression constructs are introduced into engineered cells en masse andthen protein libraries are produced from the engineered cells. Theseprotein libraries contain hundreds, thousands, or millions of individualantibodies, depending on the diversity of the starting input B cells.The resulting protein libraries are used as targeted polyclonaltherapeutics against particular pathogens, such as CMV.

Other Embodiments

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

INFORMAL SEQUENCE LISTING  Sequence 1. scFv sequence in pD912 vector CTTCAGTAATGTCTTGTTTCTTTTGTTGCAGTGGTGAGCCATTTTGACTTCGTGAAAGTTTCTTTAGAATAGTTGTTTCCAGAGGCCAAACATTCCACCCGTAGTAAAGTGCAAGCGTAGGAAGACCAAGACTGGCATAAATCAGGTATAAGTGTCGAGCACTGGCAGGTGATCTTCTGAAAGTTTCTACTAGCAGATAAGATCCAGTAGTCATGCATATGGCAACAATGTACCGTGTGGATCTAAGAACGCGTCCTACTAACCTTCGCATTCGTTGGTCCAGTTTGTTGTTATCGATCAACGTGACAAGGTTGTCGATTCCGCGTAAGCATGCATACCCAAGGACGCCTGTTGCAATTCCAAGTGAGCCAGTTCCAACAATCTTTGTAATATTAGAGCACTTCATTGTGTTGCGCTTGAAAGTAAAATGCGAACAAATTAAGAGATAATCTCGAAACCGCGACTTCAAACGCCAATATGATGTGCGGCACACAATAAGCGTTCATATCCGCTGGGTGACTTTCTCGCTTTAAAAAATTATCCGAAAAAATTTTCTAGAGTGTTGTTACTTTATACTTCCGGCTCGTATAATACGACAAGGTGTAAGGAGGACTAAACCATGGCTAAACTCACCTCTGCTGTTCCAGTCCTGACTGCTCGTGATGTTGCTGGTGCTGTTGAGTTCTGGACTGATAGACTCGGTTTCTCCCGTGACTTCGTAGAGGACGACTTTGCCGGTGTTGTACGTGACGACGTTACCCTGTTCATCTCCGCAGTTCAGGACCAGGTTGTGCCAGACAACACTCTGGCATGGGTATGGGTTCGTGGTCTGGACGAACTGTACGCTGAGTGGTCTGAGGTCGTGTCTACCAACTTCCGTGATGCATCTGGTCCAGCTATGACCGAGATCGGTGAACAGCCCTGGGGTCGTGAGTTTGCACTGCGTGATCCAGCTGGTAACTGCGTGCATTTCGTCGCAGAAGAACAGGACTAACAATTGACACCTTACGATTATTTAGAGAGTATTTATTAGTTTTATTGTATGTATACGGATGTTTTATTATCTATTTATGCCCTTATATTCTGTAACTATCCAAAAGTCCTATCTTATCAAGCCAGCAATCTATGTCCGCGAACGTCAACTAAAAATAAGCTTTTTATGCTGTTCTCTCTTTTTTTCCCTTCGGTATAATTATACCTTGCATCCACAGATTCTCCTGCCAAATTTTGCATAATCCTTTACAACATGGCTATATGGGAGCACTTAGCGCCCTCCAAAACCCATATTGCCTACGCATGTATAGGTGTTTTTTCCACAATATTTTCTCTGTGCTCTCTTTTTATTAAAGAGAAGCTCTATATCGGAGAAGCTTCTGTGGCCGTTATATTCGGCCTTATCGTGGGACCACATTGCCTGAATTGGTTTGCCCCGGAAGATTGGGGAAACTTGGATCTGATTACCTTAGCTGCAGGTACCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGGTACCCAGATCCAATTCCCGCTTTGACTGCCTGAAATCTCCATCGCCTACAATGATGACATTTGGATTTGGTTGACTCATGTTGGTATTGTGAAATAGACGCAGATCGGGAACACTGAAAAATACACAGTTATTATTCATTTAAATAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGGCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTGAAAGAATTCCGAAACGATGAGATTCCCATCTATTTTCACCGCTGTCTTGTTCGCTGCCTCCTCTGCATTGGCTGCCCCTGTTAACACTACCACTGAAGACGAGACTGCTCAAATTCCAGCTGAAGCAGTTATCGGTTACTCTGACCTTGAGGGTGATTTCGACGTCGCTGTTTTGCCTTTCTCTAACTCCACTAACAACGGTTTGTTGTTCATTAACACCACTATCGCTTCCATTGCTGCTAAGGAAGAGGGTGTCTCTCTCGAGAAAAGAGAGGCCGAAGCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGGGACAGAGTCACCATCACTTGTCGGGCAAGTCAGGGCATCAGAAATTACTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTACAGCCTGAAGATGTTGCAACTTATTACTGTCAAAGGTATAACCGTGCACCGTATACTTTTGGCCAGGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCGGCGGATCCTCTAGGTCAAGTTCCAGCGGCGGCGGTGGCAGCGGAGGCGGCGGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCCGGCAGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAATGGGTCTCAGCTATCACTTGGAATAGTGGTCACATAGACTATGCGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGATACGGCCGTATATTACTGTGCGAAAGTCTCGTACCTTAGCACCGCGTCCTCCCTTGACTATTGGGGCCAAGGTACCCTGGTCACCGTCTCGAGTGCCTCCACCAAGGGCCCATCGGTCTTCGAACAGAAGCTCATCTCAGAAGAGGATCTGTAAAGGGGCGGCCGCTCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCAGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAAACCTTCGTTTGTGCGGATC (SEQ ID NO: 1) Sequence 2. scFy Sequence 1 converted to fulllength antibody sequence in pD912 vector CTTCAGTAATGTCTTGTTTCTTTTGTTGCAGTGGTGAGCCATTTTGACTTCGTGAAAGTTTCTTTAGAATAGTTGTTTCCAGAGGCCAAACATTCCACCCGTAGTAAAGTGCAAGCGTAGGAAGACCAAGACTGGCATAAATCAGGTATAAGTGTCGAGCACTGGCAGGTGATCTTCTGAAAGTTTCTACTAGCAGATAAGATCCAGTAGTCATGCATATGGCAACAATGTACCGTGTGGATCTAAGAACGCGTCCTACTAACCTTCGCATTCGTTGGTCCAGTTTGTTGTTATCGATCAACGTGACAAGGTTGTCGATTCCGCGTAAGCATGCATACCCAAGGACGCCTGTTGCAATTCCAAGTGAGCCAGTTCCAACAATCTTTGTAATATTAGAGCACTTCATTGTGTTGCGCTTGAAAGTAAAATGCGAACAAATTAAGAGATAATCTCGAAACCGCGACTTCAAACGCCAATATGATGTGCGGCACACAATAAGCGTTCATATCCGCTGGGTGACTTTCTCGCTTTAAAAAATTATCCGAAAAAATTTTCTAGAGTGTTGTTACTTTATACTTCCGGCTCGTATAATACGACAAGGTGTAAGGAGGACTAAACCATGGCTAAACTCACCTCTGCTGTTCCAGTCCTGACTGCTCGTGATGTTGCTGGTGCTGTTGAGTTCTGGACTGATAGACTCGGTTTCTCCCGTGACTTCGTAGAGGACGACTTTGCCGGTGTTGTACGTGACGACGTTACCCTGTTCATCTCCGCAGTTCAGGACCAGGTTGTGCCAGACAACACTCTGGCATGGGTATGGGTTCGTGGTCTGGACGAACTGTACGCTGAGTGGTCTGAGGTCGTGTCTACCAACTTCCGTGATGCATCTGGTCCAGCTATGACCGAGATCGGTGAACAGCCCTGGGGTCGTGAGTTTGCACTGCGTGATCCAGCTGGTAACTGCGTGCATTTCGTCGCAGAAGAACAGGACTAACAATTGACACCTTACGATTATTTAGAGAGTATTTATTAGTTTTATTGTATGTATACGGATGTTTTATTATCTATTTATGCCCTTATATTCTGTAACTATCCAAAAGTCCTATCTTATCAAGCCAGCAATCTATGTCCGCGAACGTCAACTAAAAATAAGCTTTTTATGCTGTTCTCTCTTTTTTTCCCTTCGGTATAATTATACCTTGCATCCACAGATTCTCCTGCCAAATTTTGCATAATCCTTTACAACATGGCTATATGGGAGCACTTAGCGCCCTCCAAAACCCATATTGCCTACGCATGTATAGGTGTTTTTTCCACAATATTTTCTCTGTGCTCTCTTTTTATTAAAGAGAAGCTCTATATCGGAGAAGCTTCTGTGGCCGTTATATTCGGCCTTATCGTGGGACCACATTGCCTGAATTGGTTTGCCCCGGAAGATTGGGGAAACTTGGATCTGATTACCTTAGCTGCAGGTACCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGGTACCCAGATCCAATTCCCGCTTTGACTGCCTGAAATCTCCATCGCCTACAATGATGACATTTGGATTTGGTTGACTCATGTTGGTATTGTGAAATAGACGCAGATCGGGAACACTGAAAAATACACAGTTATTATTCATTTAAATAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGGCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTGAAAGAATTCCGAAACGATGAGATTCCCATCTATTTTCACCGCTGTCTTGTTCGCTGCCTCCTCTGCATTGGCTGCCCCTGTTAACACTACCACTGAAGACGAGACTGCTCAAATTCCAGCTGAAGCAGTTATCGGTTACTCTGACCTTGAGGGTGATTTCGACGTCGCTGTTTTGCCTTTCTCTAACTCCACTAACAACGGTTTGTTGTTCATTAACACCACTATCGCTTCCATTGCTGCTAAGGAAGAGGGTGTCTCTCTCGAGAAAAGAGAGGCCGAAGCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGGGACAGAGTCACCATCACTTGTCGGGCAAGTCAGGGCATCAGAAATTACTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTACAGCCTGAAGATGTTGCAACTTATTACTGTCAAAGGTATAACCGTGCACCGTATACTTTTGGCCAGGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAAAGGGGCGGCCGCTCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCAGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAAACCTTCGTTTGTGCGTGTTCTTTCCTGCGGTACCCAGATCCAATTCCCGCTTTGACTGCCTGAAATCTCCATCGCCTACAATGATGACATTTGGATTTGGTTGACTCATGTTGGTATTGTGAAATAGACGCAGATCGGGAACACTGAAAAATACACAGTTATTATTCATTTAAATAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGGCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTGAAAGAATTCCGAAACGATGAGATTCCCATCTATTTTCACCGCTGTCTTGTTCGCTGCCTCCTCTGCATTGGCTGCCCCTGTTAACACTACCACTGAAGACGAGACTGCTCAAATTCCAGCTGAAGCAGTTATCGGTTACTCTGACCTTGAGGGTGATTTCGACGTCGCTGTTTTGCCTTTCTCTAACTCCACTAACAACGGTTTGTTGTTCATTAACACCACTATCGCTTCCATTGCTGCTAAGGAAGAGGGTGTCTCTCTCGAGAAAAGAGAGGCCGAAGCTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCCGGCAGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAATGGGTCTCAGCTATCACTTGGAATAGTGGTCACATAGACTATGCGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGATACGGCCGTATATTACTGTGCGAAAGTCTCGTACCTTAGCACCGCGTCCTCCCTTGACTATTGGGGCCAAGGTACCCTGGTCACCGTCTCGAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAAAGGGGCGGCCGCTCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCAGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAAACCTTCGTTTGTGCGGATC (SEQ ID NO: 2)  Sequence 3. Sequence inserted into Sequence 1 tocreate Sequence 2  TTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAAAGGGGCGGCCGCTCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCAGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAAACCTTCGTTTGTGCGTGTTCTTTCCTGCGGTACCCAGATCCAATTCCCGCTTTGACTGCCTGAAATCTCCATCGCCTACAATGATGACATTTGGATTTGGTTGACTCATGTTGGTATTGTGAAATAGACGCAGATCGGGAACACTGAAAAATACACAGTTATTATTCATTTAAATAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGGCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTGAAAGAATTCCGAAACGATGAGATTCCCATCTATTTTCACCGCTGTCTTGTTCGCTGCCTCCTCTGCATTGGCTGCCCCTGTTAACACTACCACTGAAGACGAGACTGCTCAAATTCCAGCTGAAGCAGTTATCGGTTACTCTGACCTTGAGGGTGATTTCGACGTCGCTGTTTTGCCTTTCTCTAACTCCACTAACAACGGTTTGTTGTTCATTAACACCACTATCGCTTCCATTGCTGCTAAGGAAGAGGGTGTCTCTCTCGAGAAAAGAGAGGCCGAAGCT (SEQ ID NO: 3) Sequence 4. Forward primer used to linearizeSequence 1 for Gibson assembly into Sequence 2. GTCTCTCTCGAGAAAAGAGAGGCCGAAGCT SAGGTGCAGCTGGTGGAG (SEQ ID NO: 4) Sequence 5. Reverse primer used to linearizeSequence 1 for Gibson assembly into Sequence 2. CAACTGCTCATCAGATGGCGGGAAGATGAA GACAGATGGTGCAGCCACA GT (SEQ ID NO: 5) Sequence 6. Probe for capturing human heavy chain Ig CTGCCACCTGCTCTTGTCCACGGTGAGCTTGCTGT (SEQ ID NO: 6) Sequence 7. Probe for capturing human light chain Ig TGATGGGTGACTTCGCAGGCGTAGAGTTTGTGTTT (SEQ ID NO: 7) Sequence 8. Outer IgK V primer GGACTGGACATCCAGWTGACCCAGTCT (SEQ ID NO: 8) Sequence 9. Inner IgK C primer GCCGCCGCTGGAACTTGACCTAGAGGATCCGCC GACAGATGGTGCAGCC ACAGT (SEQ ID NO: 9) Sequence 10. Inner IgG V primer AGGTCAAGTTCCAGCGGCGGCGGTGGCAGCGGAGGCGGCGGT SAGGTGCAGCTGGTGGAG (SEQ ID NO: 10)  Sequence 11. Outer IgG C primer CCRYGGCTTTGTCTTGGCAT (SEQ ID NO: 11)  Sequence 12, pIC9_IgG1 AGATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGCCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAAGGATCCAAACGATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAATTCGCCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGAAGTTCGTTTGTGCAAGCTTATCGATAAGCTTTAATGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGAGCCACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGATCTATCGAATCTAAATGTAAGTTAAAATCTCTAAATAATTAAATAAGTCCCAGTTTCTCCATACGAACCTTAACAGCATTGCGGTGAGCATCTAGACCTTCAACAGCAGCCAGATCCATCACTGCTTGGCCAATATGTTTCAGTCCCTCAGGAGTTACGTCTTGTGAAGTGATGAACTTCTGGAAGGTTGCAGTGTTAACTCCGCTGTATTGACGGGCATATCCGTACGTTGGCAAAGTGTGGTTGGTACCGGAGGAGTAATCTCCACAACTCTCTGGAGAGTAGGCACCAACAAACACAGATCCAGCGTGTTGTACTTGATCAACATAAGAAGAAGCATTCTCGATTTGCAGGATCAAGTGTTCAGGAGCGTACTGATTGGACATTTCCAAAGCCTGCTCGTAGGTTGCAACCGATAGGGTTGTAGAGTGTGCAATACACTTGCGTACAATTTCAACCCTTGGCAACTGCACAGCTTGGTTGTGAACAGCATCTTCAATTCTGGCAAGCTCCTTGTCTGTCATATCGACAGCCAACAGAATCACCTGGGAATCAATACCATGTTCAGCTTGAGACAGAAGGTCTGAGGCAACGAAATCTGGATCAGCGTATTTATCAGCAATAACTAGAACTTCAGAAGGCCCAGCAGGCATGTCAATACTACACAGGGCTGATGTGTCATTTTGAACCATCATCTTGGCAGCAGTAACGAACTGGTTTCCTGGACCAAATATTTTGTCACACTTAGGAACAGTTTCTGTTCCGTAAGCCATAGCAGCTACTGCCTGGGCGCCTCCTGCTAGCACGATACACTTAGCACCAACCTTGTGGGCAACGTAGATGACTTCTGGGGTAAGGGTACCATCCTTCTTAGGTGGAGATGCAAAAACAATTTCTTTGCAACCAGCAACTTTGGCAGGAACACCCAGCATCAGGGAAGTGGAAGGCAGAATTGCGGTTCCACCAGGAATATAGAGGCCAACTTTCTCAATAGGTCTTGCAAAACGAGAGCAGACTACACCAGGGCAAGTCTCAACTTGCAACGTCTCCGTTAGTTGAGCTTCATGGAATTTCCTGACGTTATCTATAGAGAGATCAATGGCTCTCTTAACGTTATCTGGCAATTGCATAAGTTCCTCTGGGAAAGGAGCTTCTAACACAGGTGTCTTCAAAGCGACTCCATCAAACTTGGCAGTTAGTTCTAAAAGGGCTTTGTCACCATTTTGACGAACATTGTCGACAATTGGTTTGACTAATTCCATAATCTGTTCCGTTTTCTGGATAGGACGACGAAGGGCATCTTCAATTTCTTGTGAGGAGGCCTTAGAAACGTCAATTTTGCACAATTCAATACGACCTTCAGAAGGGACTTCTTTAGGTTTGGATTCTTCTTTAGGTTGTTCCTTGGTGTATCCTGGCTTGGCATCTCCTTTCCTTCTAGTGACCTTTAGGGACTTCATATCCAGGTTTCTCTCCACCTCGTCCAACGTCACACCGTACTTGGCACATCTAACTAATGCAAAATAAAATAAGTCAGCACATTCCCAGGCTATATCTTCCTTGGATTTAGCTTCTGCAAGTTCATCAGCTTCCTCCCTAATTTTAGCGTTCAACAAAACTTCGTCGTCAAATAACCGTTTGGTATAAGAACCTTCTGGAGCATTGCTCTTACGATCCCACAAGGTGGCTTCCATGGCTCTAAGACCCTTTGATTGGCCAAAACAGGAAGTGCGTTCCAAGTGACAGAAACCAACACCTGTTTGTTCAACCACAAATTTCAAGCAGTCTCCATCACAATCCAATTCGATACCCAGCAACTTTTGAGTTGCTCCAGATGTAGCACCTTTATACCACAAACCGTGACGACGAGATTGGTAGACTCCAGTTTGTGTCCTTATAGCCTCCGGAATAGACTTTTTGGACGAGTACACCAGGCCCAACGAGTAATTAGAAGAGTCAGCCACCAAAGTAGTGAATAGACCATCGGGGCGGTCAGTAGTCAAAGACGCCAACAAAATTTCACTGACAGGGAACTTTTTGACATCTTCAGAAAGTTCGTATTCAGTAGTCAATTGCCGAGCATCAATAATGGGGATTATACCAGAAGCAACAGTGGAAGTCACATCTACCAACTTTGCGGTCTCAGAAAAAGCATAAACAGTTCTACTACCGCCATTAGTGAAACTTTTCAAATCGCCCAGTGGAGAAGAAAAAGGCACAGCGATACTAGCATTAGCGGGCAAGGATGCAACTTTATCAACCAGGGTCCTATAGATAACCCTAGCGCCTGGGATCATCCTTTGGACAACTCTTTCTGCCAAATCTAGGTCCAAAATCACTTCATTGATACCATTATTGTACAACTTGAGCAAGTTGTCGATCAGCTCCTCAAATTGGTCCTCTGTAACGGATGACTCAACTTGCACATTAACTTGAAGCTCAGTCGATTGAGTGAACTTGATCAGGTTGTGCAGCTGGTCAGCAGCATAGGGAAACACGGCTTTTCCTACCAAACTCAAGGAATTATCAAACTCTGCAACACTTGCGTATGCAGGTAGCAAGGGAAATGTCATACTTGAAGTCGGACAGTGAGTGTAGTCTTGAGAAATTCTGAAGCCGTATTTTTATTATCAGTGAGTCAGTCATCAGGAGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAGTATCTATGATTGGAAGTATGGGAATGGTGATACCCGCATTCTTCAGTGTCTTGAGGTCTCCTATCAGATTATGCCCAACTAAAGCAACCGGAGGAGGAGATTTCATGGTAAATTTCTCTGACTTTTGGTCATCAGTAGACTCGAACTGTGAGACTATCTCGGTTATGACAGCAGAAATGTCCTTCTTGGAGACAGTAAATGAAGTCCCACCAATAAAGAAATCCTTGTTATCAGGAACAAACTTCTTGTTTCGAACTTTTTCGGTGCCTTGAACTATAAAATGTAGAGTGGATATGTCGGGTAGGAATGGAGCGGGCAAATGCTTACCTTCTGGACCTTCAAGAGGTATGTAGGGTTTGTAGATACTGATGCCAACTTCAGTGACAACGTTGCTATTTCGTTCAAACCATTCCGAATCCAGAGAAATCAAAGTTGTTTGTCTACTATTGATCCAAGCCAGTGCGGTCTTGAAACTGACAATAGTGTGCTCGTGTTTTGAGGTCATCTTTGTATGAATAAATCTAGTCTTTGATCTAAATAATCTTGACGAGCCAAGGCGATAAATACCCAAATCTAAAACTCTTTTAAAACGTTAAAAGGACAAGTATGTCTGCCTGTATTAAACCCCAAATCAGCTCGTAGTCTGATCCTCATCAACTTGAGGGGCACTATCTTGTTTTAGAGAAATTTGCGGAGATGCGATATCGAGAAAAAGGTACGCTGATTTTAAACGTGAAATTTATCTCAAGATCTCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTAATTCTCATGTTTGACAGCTTATCATCGATAAGCTGACTCATGTTGGTATTGTGAAATAGACGCAGATCGGGAACACTGAAAAATAACAGTTATTATTCG (SEQ ID NO: 12)  Sequence 13, Adalumimab scFv GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGGGACAGAGTCACATCACTTGTCGGGCAAGTCAGGGCATCAGAAATTACTTAGCCTGGTATCAGCAAAAACAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTACAGCTGAAGATGTTGCAACTTATTACTGTCAAAGGTATAACCGTGCACCGTATACTTTTGGCCGGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCGGCGGATCCTCTGGTCAAGTTCCAGCGGCGGCGGTGGCAGCGGAGGCGGCGGTGAGGTGCAGCTGGTGGGTCTGGGGGAGGCTTGGTACAGCCCGGCAGGTCCCTGAGACTCTCCTGTGCGGCCTCTGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGATGGGTCTCAGCTATCACTTGGAATAGTGGTCACATAGACTATGCGGACTCTGTGGAGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGATACGGCCGTATATTACTGTGCGAAAGTCTCGTACCTTAGCACCGCGTCCTCCCTTGACTATTGGGGCCAAGGTACCCTGGTCACCGTCTCGAGTGCCTCCACCAAGGGCCCATCGGTCTTC (SEQ ID NO: 13) 

The invention claimed is:
 1. A method of generating a T cell receptorlibrary, comprising: providing a plurality of circularizedpolynucleotide constructs, each comprising a first polynucleotide, asecond polynucleotide, and a linker polynucleotide linking the first andsecond polynucleotides, wherein said first polynucleotide comprises aregion encoding a first variable domain from a single isolated cell,wherein said second polynucleotide comprises a region encoding a secondvariable domain from said single isolated cell, and wherein each of saidplurality of circularized polynucleotide constructs comprises a cognatepair of linked first and second variable domains from a single isolatedcell; inserting a third polynucleotide comprising a sequence encoding apromoter, a sequence encoding a translation initiation site, or asequence encoding a constant region between said first and secondpolynucleotide in each of said plurality of circularized polynucleotideconstructs, thereby generating a plurality of recombinant T cellreceptor expression constructs; inserting said plurality of recombinantT cell receptor expression constructs into a plurality of host cells;and expressing said plurality of recombinant T cell receptor expressionconstruct in said plurality of host cells, thereby generating arecombinant T cell receptor library comprising a plurality ofrecombinant T cell receptors, wherein each of said plurality ofrecombinant T cell receptors comprises a linked beta chain variabledomain and alpha chain variable domain cognate pair from a singleisolated cell.
 2. The method of claim 1, wherein said insertion of saidthird polynucleotide is performed in parallel for said plurality ofcircularized polynucleotide constructs.
 3. The method of claim 1,wherein said first variable domain is from a T cell receptor beta chain,and said second variable domain is from a T cell receptor alpha chain.4. The method of claim 1, wherein said plurality of recombinant T cellreceptor expression constructs are expression vectors.
 5. The method ofclaim 1, wherein said insertion of said third polynucleotide comprises amethod selected from the group consisting of: Gibson assembly,site-specific digestion and ligation, and targeted recombination.